Compositions and methods for producing tobacco plants and products having altered alkaloid levels with desirable leaf quality

ABSTRACT

The present disclosure includes methods and compositions for improving leaf quality in low-alkaloid tobacco plants, e.g., by combining inducible promoters and non-coding RNAs for suppression of an ornithine decarboxylase (ODC) gene. Also provided are low alkaloid tobacco plants with normal, suppressed, or otherwise altered polyamine levels. Further provided are tobacco plants with altered total alkaloid, nicotine levels, commercially acceptable leaf grade, their development via breeding or transgenic approaches, and production of tobacco products from these tobacco plants.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/291,878, filed Mar. 4, 2019 (now U.S. Pat. No. 11,220,695), whichclaims priority to U.S. Provisional Application No. 62/638,928, filed onMar. 5, 2018, both of which are incorporated by reference herein intheir entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing contained in the ASCII file named “P34584US02_SL.txt”which is 88,060 bytes (measured in MS-Windows®) and created on Dec. 17,2021, is filed electronically herewith and incorporated by reference inits entirety.

FIELD

The present disclosure includes tobacco plants having altered totalalkaloid and nicotine levels and commercially acceptable leaf grade,their development via breeding or transgenic approaches, and productionof tobacco products from these tobacco plants.

BACKGROUND

Tobacco is one of the most widely grown non-food crops in the world withglobal production exceeding 7.4 million tons (FAOSTAT, Food andAgriculture Organization of the United Nations (FAO) (2014),faostat.fao.org) and resulting tobacco products having an annual globalmarket size of USD 770 billion (Euromonitor International, 2016).Nicotine is the main alkaloid accumulating in tobacco leaves. Nicotineand other minor alkaloids are also precursors to tobacco-specificnitrosamines (TSNA). Demands exist for development of tobacco cultivarswith lower levels of nicotine.

In commercial tobacco cultivars, nicotine represents 90-95% of the totalalkaloid pool or 2-5% of total leaf dry weight (Saitoh F, Nona M,Kawashima N (1985). The alkaloid contents of sixty Nicotiana species.Phytochem. 24: 477-480). Nicotine is synthesized in the roots (Dawson RF(1942) Accumulation of nicotine in reciprocal grafts of tomato andtobacco. Am. J. Bot. 29: 66-71), and translocated through the xylem(Baldwin IT (1988). The alkaloidal responses of wild tobacco to real andsimulated herbivory. Oecologia 77: 378-381) to aerial parts of the plant(Hildreth S B, Gehman E A, Yang H, Lu R H, Ritesh K C, Harich K C, Yu S,Lin J, Sandoe J L, Okumoto S, Murphyd, A S, Jeleskoaet J G (2011).Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism.Proc. Natl. Acad. Sci. USA 108: 18179-18184) where it accumulates in theleaves and is exuded by trichomes in response to insect herbivory(Kessler A, Baldwin IT (2002). Plant responses to insect herbivory: theemerging molecular analysis. Annu Rev. Plant Biol. 53: 299-328).Nicotine biosynthesis is influenced by genetic factors, plantdevelopment, biotic and abiotic stresses, phytohormonal signals andagronomic management practices such as topping and suckering (Wang S S,Shi Q M, Li W Q, Niu J F, Li C J, Zhang F S (2008). Nicotineconcentration in leaves of flue-cured tobacco plants as affected byremoval of the shoot apex and lateral buds. J. Integr. Plant Bio. 50:958-964; Shoji T, Hashimoto T (2015). Stress-induced expression ofNICOTINE2-locus genes and their homologs encoding Ethylene ResponseFactor transcription factors in tobacco. Phytochem. 113: 41-49). Thegenetic regulation of nicotine biosynthesis correlates to twoindependent loci, Nic1 and Nic2, which have a synergistic effect onnicotine levels, but the effect of Nic1 is ˜2.4 times stronger than thatof Nic2 (Legg P D, Collins G B (1971). Inheritance of percent totalalkaloids in Nicotiana tabacum L. II. Genetic effects of two loci inBurley 21×LA Burley 21 populations. Can. J. Genet. Cytol. 13: 287-291).Both loci also influence the expression of numerous other genesunrelated to the nicotine biosynthesis pathway (Kidd S K, Melillo A A,Lu R H, Reed D G, Kuno N, Uchida K, Furuya M, Jelesko J G (2006). The Aand B loci in tobacco regulate a network of stress response genes, fewof which are associated with nicotine biosynthesis. Plant Mol. Biol. 60:699-716; Shoji T, Kajikawa M, Hashimoto T (2010). Clusteredtranscription factor genes regulate nicotine biosynthesis in tobacco.Plant Cell 22: 3390-3409). Transcriptional analysis has shown that theNic2 locus is a gene cluster that encodes at least seven ethyleneresponse transcription factors (ERFs) (Shoji et al. 2010).

Homozygous mutations of either one or both loci can be used to createnear-isogenic Burley 21 lines with reduced alkaloid levels, i.e. ahigh-intermediate (HI) variety with the genotype nic2, alow-intermediate (LI) variety with the genotype nic1, and a low-alkaloid(LA) variety with the genotype nic1nic2 (Legg P D, Chaplin J F, CollinsG B (1969). Inheritance of percent total alkaloids in Nicotiana tabacumL. J. Hered. 60: 213-217; Legg et al. 1971). LA Burley 21 plants containonly ˜5.7% of the total alkaloid levels found in the normal-alkaloid(NA) wild-type variety (Legg P D, Collins G B, Littion C C (1970).Registration of LA Burley 21 tobacco germplasm. Crop. Sci. 10: 212). InLA plants, the synergistic effect of the nic1 and nic2 mutations alsocauses an unfavorable leaf phenotype characterized by lower yields,delayed ripening and senescence, higher susceptibility to insectherbivory, and poor end-product quality after curing (Chaplin J F, WeeksW W (1976). Association between percent total alkaloids and other traitsin flue-cured tobacco. Crop Sci. 16: 416-418; Legg et al. 1970; ChaplinJ F, Burk L G (1983). Agronomic, chemical, and smoke characteristics offlue-cured tobacco lines with different levels of total alkaloids. CropSci. 75: 133-136).

There is a need to identify genes that restore unfavorable leafphenotypes in the LA variety of tobacco plants, and to develop tobaccoplants and products that contain altered nicotine levels (e.g., reducednicotine) while maintaining (if not making superior) tobacco leafquality.

SUMMARY

In an aspect, the present disclosure provide a tobacco plant comprisingan inducible promoter operably linked to a transcribable DNA sequenceencoding a non-coding RNA for suppression of an ornithine decarboxylase(ODC) gene.

In another aspect, the present disclosure provide a tobacco plant, orpart thereof, comprising relative to a control tobacco plant: a firstgenome modification providing a lower level of nicotine or totalalkaloid, and a second genome modification providing a comparable levelof one or more traits selected from the group consisting of total leafpolyamine level, total root polyamine level, total leaf chlorophylllevel, mesophyll cell number per leaf area unit, and leaf epidermal cellsize; and wherein said control plant does not have both said first andsaid second genome modifications.

In an aspect, the present disclosure provide a method for improving leafquality in a reduced-alkaloid tobacco plant, said method comprising:growing a tobacco plant, reducing the level of putrescine in saidtobacco plant, harvesting leaves from said tobacco plant.

In another aspect, the present disclosure provide a method for improvingleaf quality in a reduced-alkaloid tobacco plant, said methodcomprising: growing a tobacco plant, suppressing the expression oractivity of an ornithine decarboxylase (ODC) gene in said tobacco plant,harvesting leaves from said tobacco plant

In an aspect, a tobacco plant is provided having suppressed MYB8activity via either transgene suppression, mutagenesis, or targetedgenome editing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Phenotypic characterization of N. tabacum L. cv. Burley 21normal-alkaloid (NA) wild-type plants and three mutant varieties grownin the greenhouse. FIG. 1A: Total chlorophyll content of wild-type (NA)high-intermediate (HI, nic2), low-intermediate (LI, nic1) andlow-alkaloid (LA, nic1nic2) lines grown in the greenhouse. Chlorophyllwas measured twice per leaf in all leaves longer than 15 cm at differentdevelopmental stages: before flowering (2.5 weeks before topping), attopping, 1 week post topping (WPT), 2.5 WPT and harvest. Values aremeans of four biological replicates. FIG. 1B: Representative photos ofNA and LA plants at harvest. FIG. 1C: Time-course evaluation ofmesophyll cell number in leaf 15. FIG. 1D: Microscopic images ofmesophyll cells of leaf 15 from NA and LA plants at different plantdevelopmental stages. Bar=100 μm. Values in A and C are means of sixbiological replicates. Error bars represent standard deviations of themean. Statistical difference to NA is shown: *p<0.05.

FIG. 2 : Polyamine analysis in field and greenhouse grown NA and LAplants. FIG. 2A: Free (F) and conjugated (C) putrescine (Put),spermidine (Spd) and spermine (Spm) content from five well-expandedupper leaves of NA and LA plants (three biological replicates grown inthe field) 1 week after topping. FIG. 2B: Time-course monitoring oftotal polyamine content in NA and LA plants grown in the greenhouse.Leaf samples were collected from leaves at the same stage ofdevelopment: before flowering (leaf 12), at topping (leaf 19) and atharvest leaf (24). Root samples were collected at topping and harvest.Values are means of three (A)/four (B) biological replicates. Error barsrepresent standard deviations of the mean. Statistical difference to NAis shown: *p<0.05; **p<0.005. PA: polyamine; FW: fresh weight.

FIG. 3 : Polyamine content in the leaves and roots of NA, HI, LI and LAplants grown in the greenhouse. Free (F) and conjugated (C) putrescine(Put), spermidine (Spd) and spermine (Spm) fractions in leaves (FIG. 3A)and roots (FIG. 3B) of NA, HI, LI and LA plants before flowering (leaf6), at topping (young leaf 23, roots) and at harvest (matured leaf 23,roots) are shown. Samples were collected 4 h after illumination, frozenimmediately in liquid nitrogen and analyzed by LC-MS/MS. Values aremeans of three biological replicates. Error bars represent standarddeviations of the mean. Statistical difference is shown: *p<0.05;**p<0.001, indicating that the LI and LA plants are significantlydifferent from NA plants under the same conditions. Only samples fromtopping and harvest were available for roots. FW: fresh weight.

FIG. 4 : Activity of polyamine biosynthesis enzymes. Analysis ofarginine decarboxylase (ADC) (FIG. 4A) and ornithine decarboxylase (ODC)(FIG. 4B) activity in leaves and roots of NA and LA plants at topping(young leaf 23, roots) and harvest (matured leaf 23, roots). Values aremeans of three biological replicates. Error bars represent standarddeviations of the mean. Statistical difference to NA is shown: *p<0.05;**p<0.001.

FIG. 5 : Representative photos of leaf 23 from untreated NA and LAplants and LA plants treated with polyamine biosynthesis inhibitorsand/or plant growth regulator at harvest. D-arginine (5 mM) is aninhibitor of ADC; DFMO (difluoromethylornithine, 2 mM) is an inhibitorof ODC, ETH (Ethephon®, 0.5 mM) is a growth regulator.

FIG. 6 : Treatment of LA plants with polyamine biosynthesis inhibitorsand/or Ethephon®. Comparative total, free and conjugated polyamine inleaves (FIG. 6A) and roots (FIG. 6B) of untreated and treated LA plantsat topping and harvest. Tobacco plants were grown in the greenhouse inthe absence (NA and LA) or presence (LA) of polyamine biosynthesisinhibitors and/or Ethephon® (5 mM D-arginine, 2 mM DFMO, 2 mM DFMO/0.5mM Ethephon® or 0.5 mM Ethephon® alone). D-arginine and DFMO wereapplied three times per week from before flowering to harvest for aperiod of 6 weeks, whereas Ethephon® treatment started after topping(2.5 weeks later) until harvest. Samples were collected 4 h afterillumination from leaf 23 or roots of four biological replicates pergenotype or treatment. The fold change between the mean polyaminecontent from untreated LA plants (gray bars), or plants treated withD-arginine (black bars), DFMO (white bars), DFMO/Ethephon® (horizontallined bars), and Ethephon® (divot bars) are plotted. Error barsrepresent standard deviations of the mean (n=4). Statistical differenceto the mean of LA/NA is indicated: *p<0.05. The red line representspolyamine content in NA.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID Nos: 1 to 11 set forth sequences of exemplary promoters fortopping responsive root specific or preferred expression.

SEQ ID Nos: 11 to 21 set forth sequences of exemplary promoters fortopping responsive leaf specific or preferred expression.

SEQ ID No: 22 sets forth a sequence of an exemplary DNA constructencoding a non-coding RNA suppressing an ornithine decarboxylase (ODC).

SEQ ID Nos: 23 to 28 set forth cDNA sequences of exemplary tobacco ODCgenes.

SEQ ID Nos: 29 to 34 set forth amino acid sequences encoded by exemplaryODC genes.

SEQ ID Nos: 35 and 36 set forth two miRNA sequences targeting an ODCgene in accordance with the present disclosure.

Various sequences include “N” in nucleotide sequences or “X” in aminoacid sequences. “N” can be any nucleotide, e.g., A, T, G, C, or adeletion or insertion of one or more nucleotides. In some instant, astring of “N” are shown. The number of “N” does not necessarilycorrelate with the actual number of undetermined nucleotides at thatposition. The actual nucleotide sequences can be longer or shorter thanthe shown segment of “N”. Similarly, “X” can be any amino acid residueor a deletion or insertion of one or more amino acids. Again, the numberof “X” does not necessarily correlate with the actual number ofundetermined amino acids at that position. The actual amino acidsequences can be longer or shorter than the shown segment of “X”.Notwithstanding the use of A, T, G, C (compared to A, U, G, C) indescribing any SEQ ID in the sequence listing, that SEQ ID can alsorefer to a RNA sequence, depending on the context in which the SEQ ID ismentioned.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. One skilled in the art will recognize many methods can be usedin the practice of the present disclosure. Indeed, the presentdisclosure is in no way limited to the methods and materials described.For purposes of the present disclosure, the following terms are definedbelow.

Any references cited herein, including, e.g., all patents andpublications are incorporated by reference in their entirety.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. To avoid any doubt, usedherein, terms or phrases such as “about”, “at least”, “at least about”,“at most”, “less than”, “greater than”, “within” or alike, when followedby a series of list of numbers of percentages, such terms or phrases aredeemed to modify each and every number of percentage in the series orlist.

As used herein, a tobacco plant can be from any plant from the Nicotianagenus including, but not limited to Nicotiana tabacum, Nicotianaamplexicaulis PI 271989; Nicotiana benthamiana PI 555478; Nicotianabigelovii PI 555485; Nicotiana debneyi; Nicotiana excelsior PI 224063;Nicotiana glutinosa PI 555507; Nicotiana goodspeedii PI 241012;Nicotiana gossei PI 230953; Nicotiana hesperis PI 271991; Nicotianaknightiana PI 555527; Nicotiana maritima PI 555535; Nicotianamegalosiphon PI 555536; Nicotiana nudicaulis PI 555540; Nicotianapaniculata PI 555545; Nicotiana plumbaginifolia PI 555548; Nicotianarepanda PI 555552; Nicotiana rustica; Nicotiana suaveolens PI 230960;Nicotiana sylvestris PI 555569; Nicotiana tomentosa PI 266379; Nicotianatomentosiformis; and Nicotiana trigonophylla PI 555572.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising an inducible promoter operably linked to atranscribable DNA sequence encoding a non-coding RNA for suppression ofan ornithine decarboxylase (ODC) gene. In one aspect, tobacco plantscomprise a mutation or a transgene conferring a reduced level ofnicotine. In an aspect, tobacco plants are low-alkaloid tobacco plants.In one aspect, tobacco plants of the present disclosure comprise a nic1mutation, a nic2 mutation, or both. In an aspect, tobacco plantscomprise nicotine at a level below 1%, below 2%, below 5%, below 8%,below 10%, below 12%, below 15%, below 20%, below 25%, below 30%, below40%, below 50%, below 60%, below 70%, or below 80% of the nicotine levelof a control plant when grown in similar growth conditions, where thecontrol plant shares an essentially identical genetic background withthe tobacco plant except a low-nicotine conferring mutation ortransgene. In another aspect, tobacco plants comprise nicotine or totalalkaloids at a level below 1%, below 2%, below 5%, below 8%, below 10%,below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below50%, below 60%, below 70%, or below 80% of the nicotine or totalalkaloids level of the control plant when grown in similar growthconditions. In another aspect, tobacco plants comprise a total alkaloidlevel selected from the group consisting of less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%,less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05% ofthe nicotine level of a control plant when grown in similar growthconditions, where the control plant shares an essentially identicalgenetic background with the tobacco plant except a low-nicotineconferring mutation or transgene. In another aspect, tobacco plantscomprise a nicotine or total alkaloid level selected from the groupconsisting of less than 3%, less than 2.75%, less than 2.5%, less than2.25%, less than 2.0%, less than 1.75%, less than 1.5%, less than 1.25%,less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%,less than 0.1%, and less than 0.05% of the nicotine or total alkaloidslevel of the control plant when grown in similar growth conditions.

In an aspect, tobacco plants comprise a transgene or mutation directlysuppressing the expression or activity of one or more, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, eleven or more, twelve ormore, thirteen or more, fourteen or more, fifteen or more, sixteen ormore, seventeen or more, eighteen or more, nineteen or more, twenty ormore, or all twenty-one genes or loci encoding a protein selected fromthe group consisting of aspartate oxidase, agmatine deiminase (AIC),arginase, diamine oxidase, arginine decarboxylase (ADC),methylputrescine oxidase (MPO), NADH dehydrogenase, ornithinedecarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1,BBL, MYC2, nic1, nic2, ethylene response factor (ERF) transcriptionfactor, nicotine uptake permease (NUP), and MATE transporter. See Deweyand Xie, Molecular genetics of alkaloid biosynthesis in Nicotianatabacum, Phytochemistry 94 (2013) 10-27.

In an aspect, tobacco plants further comprise one or more mutations inone or more, two or more, three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or all ten genesselected from the group consisting of ERF32, ERF34, ERF39, ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect,tobacco plants further comprise one or more mutations in ERF189, ERF115,or both. In an aspect, tobacco plants further comprise one or moretransgenes targeting and suppressing a gene encoding one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or all ten proteins selected from thegroup consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104,ERF179, ERF17, and ERF168.

In an aspect, tobacco plants are capable of producing leaves, whencured, having a USDA grade index value selected from the groupconsisting of 55 or more, 60 or more, 65 or more, 70 or more, 75 ormore, 80 or more, 85 or more, 90 or more, and 95 or more. In anotheraspect, tobacco plants are capable of producing leaves, when cured,having a USDA grade index value comparable to that of a control plantwhen grown and cured in similar conditions, where the control plantshares an essentially identical genetic background with the tobaccoplant except a low-nicotine conferring mutation or transgene. In afurther aspect, tobacco plants are capable of producing leaves, whencured, having a USDA grade index value of at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 98% of theUSDA grade index value of a control plant when grown in similarconditions, where the control plant shares an essentially identicalgenetic background with the tobacco plant except a low-nicotineconferring mutation or transgene. In a further aspect, tobacco plantsare capable of producing leaves, when cured, having a USDA grade indexvalue of between 65% and 130%, between 70% and 130%, between 75% and130%, between 80% and 130%, between 85% and 130%, between 90% and 130%,between 95% and 130%, between 100% and 130%, between 105% and 130%,between 110% and 130%, between 115% and 130%, or between 120% and 130%of the USDA grade index value of the control plant. In a further aspect,tobacco plants are capable of producing leaves, when cured, having aUSDA grade index value of between 70% and 125%, between 75% and 120%,between 80% and 115%, between 85% and 110%, or between 90% and 100% ofthe USDA grade index value of the control plant.

In another aspect, tobacco plants are capable of producing leaves, whencured, having a USDA grade index value selected from the groupconsisting of 55 or more, 60 or more, 65 or more, 70 or more, 75 ormore, 80 or more, 85 or more, 90 or more, and 95 or more. In anotheraspect, tobacco plants are capable of producing leaves, when cured,having a USDA grade index value selected from the group consisting ofbetween 50 and 95, between 55 and 95, between 60 and 95, between 65 and95, between 70 and 95, between 75 and 95, between 80 and 95, between 85and 95, between 90 and 95, between 55 and 90, between 60 and 85, between65 and 80, between 70 and 75, between 50 and 55, between 55 and 60,between 60 and 65, between 65 and 70, between 70 and 75, between 75 and80, between 80 and 85, between 85 and 90, and between 90 and 95. In afurther aspect, tobacco plants are capable of producing leaves, whencured, having a USDA grade index value of at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 98% of theUSDA grade index value of the control plant. In a further aspect,tobacco plants are capable of producing leaves, when cured, having aUSDA grade index value of between 65% and 130%, between 70% and 130%,between 75% and 130%, between 80% and 130%, between 85% and 130%,between 90% and 130%, between 95% and 130%, between 100% and 130%,between 105% and 130%, between 110% and 130%, between 115% and 130%, orbetween 120% and 130% of the USDA grade index value of the controlplant. In a further aspect, tobacco plants are capable of producingleaves, when cured, having a USDA grade index value of between 70% and125%, between 75% and 120%, between 80% and 115%, between 85% and 110%,or between 90% and 100% of the USDA grade index value of the controlplant.

In an aspect, the present disclosure also provides a tobacco variety,cultivar, or line comprising a mutation selected from the groupconsisting of a nic1 mutation, a nic2 mutation, and a combinationthereof, where the tobacco variety, cultivar, or line has a leaf gradecomparable to the leaf grade of a control tobacco variety, cultivar, orline when grown in similar growth conditions, where the control tobaccovariety shares an essentially identical genetic background with thetobacco variety, cultivar, or line except the mutation.

In an aspect, the present disclosure further provides non-transgenictobacco plants, or part thereof, comprising a nicotine or total alkaloidlevel selected from the group consisting of less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%,less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%,where the tobacco plants are capable of producing leaves, when cured,having a USDA grade index value of 50 or more 55 or more, 60 or more, 65or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, and95 or more. In another aspect, such non-transgenic tobacco plantscomprise a nicotine level of less than 2.0% and are capable of producingleaves, when cured, having a USDA grade index value of 70 or more. In afurther aspect, such non-transgenic tobacco plants comprise a nicotinelevel of less than 1.0% and are capable of producing leaves, when cured,having a USDA grade index value of 70 or more.

In an aspect, the present disclosure also provides a tobacco plant, orpart thereof, comprising a non-transgenic mutation, where thenon-transgenic mutation reduces the nicotine or total alkaloid level ofthe tobacco plant to below 1%, below 2%, below 5%, below 8%, below 10%,below 12%, below 15%, below 20%, below 25%, below 30%, below 40%, below50%, below 60%, below 70%, or below 80% of the nicotine level of acontrol plant when grown in similar growth conditions, where the tobaccoplant is capable of producing leaves, when cured, having a USDA gradeindex value comparable to the USDA grade index value of the controlplant, and where the control plant shares an essentially identicalgenetic background with the tobacco plant except the non-transgenicmutation.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a mutation in a gene or locus, where the mutation isabsent from LA Burley 21. In an aspect, tobacco plants provided hereincomprise a shorter chromosomal introgression at a locus of interestcompared to LA Burley 21. In another aspect, tobacco plants providedherein comprise no deletion of a complete gene or a complete geniccoding sequence in the locus of interest. In an aspect, tobacco plantsprovided herein are homozygous at the locus of interest. In anotheraspect, tobacco plants provided herein are heterozygous at the locus ofinterest. In an aspect, tobacco plants provided herein comprise amutation selected from the group consisting of a point mutation, adeletion, an insertion, a duplication, and an inversion at the gene orlocus of interest. In an aspect, mutations in the tobacco plantsprovided herein are introduced by an approach selected from the groupconsisting of random mutagenesis and targeted mutagenesis. In anotheraspect, mutations in the tobacco plants provided herein are introducedby a targeted mutagenesis approach selected from the group consisting ofmeganuclease, zinc finger nuclease, TALEN, and CRISPR.

As used herein, a mutation refers to an inheritable genetic modificationintroduced into a gene to alter the expression or activity of a productencoded by the gene. Such a modification can be in any sequence regionof a gene, for example, in a promoter, 5′ UTR, exon, intron, 3′ UTR, orterminator region. In an aspect, a mutation reduces, inhibits, oreliminates the expression or activity of a gene product. In anotheraspect, a mutation increases, elevates, strengthens, or augments theexpression or activity of a gene product. In an aspect, mutations arenot natural polymorphisms that exist in a particular tobacco variety orcultivar. As used herein, a “mutant allele” refers to an allele from alocus where the allele comprises a mutation. As used herein, “mutagenic”refers to generating a mutation without involving a transgene or with nomutation-related transgene remaining in an eventual mutant. In anaspect, mutagenic is cisgenic. In another aspect, mutagenic is via geneor genome editing. In a further aspect, mutagenic is via randommutagenesis, for example, chemical (e.g., EMS) or physical(r-irradiation) mutagenesis.

In an aspect, tobacco plants provided herein comprise one or moremutations within one or more genes comprising a coding sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or 100% identity to a sequence selected fromthe group consisting of SEQ ID NOs: 23 to 28, and fragments thereof. Inan aspect, one or more mutations reduce the expression or activity ofone or more genes comprising a coding sequence having at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, or 100% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 23 to 28, and fragments thereof.

In an aspect, tobacco plants provided herein comprise one or moremutations within one or more genes encoding a polypeptide having atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, or 100% identity to a sequence selected fromthe group consisting of SEQ ID NOs: 29 to 34, and fragments thereof. Inan aspect, one or more mutations reduce the expression or activity ofone or more genes encoding a polypeptide having at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%, or 100% identity to a sequence selected from the group consistingof SEQ ID NOs: 29 to 34, and fragments thereof.

LA Burley 21 (also referenced as LA BU21) is a low total alkaloidtobacco line produced by incorporation of a low alkaloid gene(s) from aCuban cigar variety into Burley 21 through several backcrosses (Legg etal. 1970). It has approximately 0.2% total alkaloids (dry weight)compared to the about 3.5% (dry weight) of its parent, Burley 21. LABU21 has a leaf grade well below commercially acceptable standards. LABU21 also exhibits other unfavorable leaf phenotypes characterized bylower yields, delayed ripening and senescence, higher susceptibility toinsect herbivory, and poor end-product quality after curing (Chaplin andWeeks, 1976; Legg et al. 1970; Chaplin and Burk 1983). LA BU21 leavesfurther exhibit traits such as higher polyamine content, higherchlorophyll content and more mesophyll cells per unit leaf area.

In plants, polyamines are reportedly involved in developmental,physiological and metabolic processes such as cell growth and division,stress tolerance, vascular differentiation, lignin polymerization,pathogen defense, senescence and ripening (Fariduddin Q, Varshney P,Yusuf M, Ahmad A (2013) Polyamines: potent modulators of plant responsesto stress. J. Plant Interac. 8: 1-16; Kusano T, Suzuki H (2015).Polyamines a universal molecular nexus for growth, survival andspecialized metabolism. Tokyo: Springer). Several studies have linkedpolyamines to the regulation of plant cell senescence(Sobieszczuk-Nowicka, E., Kubala, S., Zmienko, A., Malecka, A., Legocka,J. 2016. From accumulation to degradation: Reprograming polyaminemetabolism facilitates dark-induced senescence in Barley leaf cells.Front. Plant Sci. doi: 10.3389/fpls.2015.01198). In fruit and vegetativetissues, polyamines act as anti-senescence and anti-ripening regulatorsthat prevent the decay of chloroplast photosystem complexes and changesin cell wall/membrane composition (Lester G E (2000). Polyamines andtheir cellular anti-senescence properties in honey dew musk melon fruit.Plant Sci. 160: 105-112; Mattoo A K, Handa A K (2008). Higher polyaminesrestore and enhance metabolic memory in ripening fruit. Plant Sci. 174:386-393; Serafini-Fracassini D, Di Sandro A, Del Duca S (2010). Sperminedelays leaf senescence in Lactuca sativa and prevents the decay ofchloroplast photosystems. Plant Physiol. Biochem. 48: 602-611). Higherlevels of polyamines increase the longevity of tomato vines (Mehta R A,Cassol T, Li N, Ali N, Handa A K, Mattoo A K (2002). Engineeredpolyamine accumulation in tomato enhances phytonutrient content, juicequality and vine life. Nat. Biotechnol. 20: 613-618), and delayedripening and leaf senescence was observed in transgenic tomato plantsoverexpressing a yeast spermidine synthase (Nambeesan S, Datsenka T,Ferruzzi M G, Malladi A, Mattoo A K, Handa A K (2010). Overexpression ofyeast spermidine synthase impacts ripening, senescence and decaysymptoms in tomato. Plant J. 63: 836-847). Polyamines may act directlyby stabilizing cell walls or through crosstalk with phytohormones suchas ethylene, abscisic acid, cytokinins and gibberellins (Kussano andSuzuki 2015).

In most plants, putrescine can be synthesized either directly fromornithine by ornithine decarboxylase (ODC) or from arginine via threeenzymatic steps, initiated by arginine decarboxylase (ADC) (Michael A J,Furze J M, Rhodes M J, Burtin D (1996). Molecular cloning and functionalidentification of a plant ornithine decarboxylase cDNA. Biochem. J. 314:241-248; Piotrowski M, Janowitz T, Kneifel H (2003). Plant C—Nhydrolases and the identification of a plant N-carbamoylputrescineamidohydrolase involved in polyamine biosynthesis J. Biol. Chem. 278:1708-1712; Illingworth C, Mayer M J, Elliot K, Hanfrey C, Walton N J,Michael A J (2003). The diverse bacterial origins of the Arabidopsispolyamine biosynthetic pathway FEBS Letters 549: 26-30). Previousstudies have stated that the ADC route to putrescine has only a minoreffect on the alkaloid profile of tobacco whereas the ODC pathway playsthe major role in nicotine biosynthesis (Chintapakorn Y, Hamill J D(2007). Antisense-mediated reduction in ADC activity causes minoralterations in the alkaloid profile of cultured hairy root andregenerated transgenic plants of Nicotiana tabacum. Phytochem. 68:2465-2479; DeBoer K D, Dalton H L, Edward F J, Hamill J D (2011).RNAi-mediated down-regulation of ornithine decarboxylase (ODC) leads toreduced nicotine and increased anatabine levels in transgenic Nicotianatabacum L. Phytochem. 72: 344-355; DeBoer K D, Dalton H L, Edward F J,Ryan S M, Hamill J D (2013). RNAi-mediated down-regulation of ornithinedecarboxylase (ODC) impedes wound-stress stimulation of anabasinesynthesis in Nicotiana glauca. Phytochem. 86: 21-28; Dalton H L,Blomstedt C K, Neale A D, Gleadow R, DeBoer K D, Hamill J D (2016).Effects of down-regulating ornithine decarboxylase uponputrescine-associated metabolism and growth in Nicotiana tabacum L. J.Exp. Bot. 67: 3367-3381). Putrescine is converted to spermidine and thenspermine by the successive addition of aminopropyl groups derived fromdecarboxylated S-adenosylmethionine (SAM), in reactions catalyzed by theenzymes spermidine synthase and spermine synthase, respectively. SAM isalso a substrate for the biosynthesis of ethylene (Tiburcio A F,Altabella T, Bitrián M, Alcazar R (2014). The roles of polyamines duringthe lifespan of plants: from development to stress. Planta 240: 1-18),which regulates senescence and fruit ripening (Fluhr R, Mattoo A K(1996). Ethylene—biosynthesis and perception. Crit. Rev. Plant Sci.15:479-523). The polyamine and ethylene biosynthesis pathways competefor the common precursor SAM but have opposing developmental effects,particularly during the developmental switch from vegetative growth toripening/senescence (Nambeesan S, Handa A K, Mattoo A K (2008).Polyamines and regulation of ripening and senescence. In: Paliyath G,Murr D P, Handa A K, Lurie S (eds) Postharvest biology and technology offruits, vegetables and flowers. Willey-Blackwell Publ, Ames. pp 319-340,Harpaz-Saad S, Yoon G M, Mattoo A K, Kieber J J (2012). The formation ofACC and competition between polyamines and ethylene for SAM. Annu. PlantReviews. 44: 53-81, Gupta A, Pal R K, Rajam M V (2013). Delayed ripeningand improved fruit processing quality in tomato by RNAi-mediatedsilencing of three homologs of 1-aminopropane-1-carboxylate synthasegene. J. Plant Physiol. 170: 987-995). Polyamine levels decrease andethylene levels increase during the onset of fruit ripening in tomato(Saftner R A, Baldi B G (1990). Polyamine levels and tomato fruitdevelopment: possible interaction with ethylene. Plant Physiol. 92:547-550; Morilla A, Garcia J M, Albi M A (1996). Free polyamine contentsand decarboxylase activities during tomato development and ripening. J.Agri. Food Chem. 44: 2608-2611) and avocado (Kushad M M, Yelenosky G,Knight R (1988). Interrelationship of polyamine and ethylenebiosynthesis during avocado fruit development and ripening. PlantPhysiol. 87:463-467), which reflects the mutually antagonistic effect ofethylene on polyamine biosynthesis and vice versa (Harpaz-Saad et al.2012; Anwar R, Mattoo A, Handa A (2015). Polyamine interactions withplant hormones: crosstalk at several levels in Kusano T, Suzuki H (eds).Polyamines a Universal Molecular Nexus for Growth, Survival andSpecialized Metabolism. Tokyo: Springer. pp 267-303). However,transgenic tomato plants expressing yeast S-adenosylmethioninedecarboxylase (SAMDC) under the control of the ripening-specific E8promoter produced higher levels of ethylene and polyaminessimultaneously during fruit ripening, indicating the absence of anycompetition for SAM in this system (Mehta R A, Cassol T, Li N, Ali N,Handa A K, Mattoo A K (2002). Engineered polyamine accumulation intomato enhances phytonutrient content, juice quality and vine life. Nat.Biotechnol. 20: 613-618).

Without being bound to any scientific theory, the suppression ofnicotine biosynthesis in LA tobacco plants can affect crosstalk betweenthe nicotine, polyamine and ethylene pathways, resulting in theaccumulation of putrescine. This would in turn increase metabolic fluxtowards the higher polyamines spermidine and spermine while inhibitingethylene biosynthesis, causing a dramatic effect on leaf ripening andsenescence.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and capable of producing a leaf comprising a comparablelevel of one or more polyamines relative to a comparable leaf of acontrol plant not comprising the same mutation or transgene. In oneaspect, a comparable level of one or more polyamines is within 20%,17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in acomparable leaf of a control plant not comprising the same mutation ortransgene. In an aspect, a comparable level of one or more polyamines isbetween 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3%and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%,between 12% and 13%, between 13% and 14%, between 14% and 15%, between15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and19%, or between 19% and 20% of the level in a comparable leaf of acontrol plant not comprising the same mutation or transgene. In afurther aspect, a comparable level of one or more polyamines is between0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level ina comparable leaf of a control plant not comprising the same mutation ortransgene.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and capable of producing a leaf comprising a comparablechlorophyll level relative to a comparable leaf of a control plant notcomprising the same mutation or transgene. In one aspect, a comparablechlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%,or 1% of the level in a comparable leaf of a control plant notcomprising the same mutation or transgene. In an aspect, a comparablechlorophyll level is between 0.5% and 1%, between 1% and 2%, between 2%and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%,between 11% and 12%, between 12% and 13%, between 13% and 14%, between14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and18%, between 18% and 19%, or between 19% and 20% of the level in acomparable leaf of a control plant not comprising the same mutation ortransgene. In a further aspect, a comparable chlorophyll level isbetween 0.5% and 5%, between 5% and 10%, or between 10% and 20% of thelevel in a comparable leaf of a control plant not comprising the samemutation or transgene.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and capable of producing a leaf comprising a comparablenumber of mesophyll cell per unit of leaf area relative to a comparableleaf of a control plant not comprising the same mutation or transgene.In one aspect, a comparable number of mesophyll cell per unit of leafarea is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of thelevel in a comparable leaf of a control plant not comprising the samemutation or transgene. In an aspect, a comparable number of mesophyllcell per unit of leaf area is between 0.5% and 1%, between 1% and 2%,between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9%and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%,between 14% and 15%, between 15% and 16%, between 16% and 17%, between17% and 18%, between 18% and 19%, or between 19% and 20% of the level ina comparable leaf of a control plant not comprising the same mutation ortransgene. In a further aspect, a comparable number of mesophyll cellper unit of leaf area is between 0.5% and 5%, between 5% and 10%, orbetween 10% and 20% of the level in a comparable leaf of a control plantnot comprising the same mutation or transgene.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and capable of producing a leaf comprising a comparableepidermal cell size relative to a comparable leaf of a control plant notcomprising the same mutation or transgene. In one aspect, a comparableepidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%,2.5%, or 1% of the level in a comparable leaf of a control plant notcomprising the same mutation or transgene. In an aspect, a comparableepidermal cell size is between 0.5% and 1%, between 1% and 2%, between2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%,between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and10%, between 11% and 12%, between 12% and 13%, between 13% and 14%,between 14% and 15%, between 15% and 16%, between 16% and 17%, between17% and 18%, between 18% and 19%, or between 19% and 20% of the level ina comparable leaf of a control plant not comprising the same mutation ortransgene. In a further aspect, a comparable epidermal cell size isbetween 0.5% and 5%, between 5% and 10%, or between 10% and 20% of thelevel in a comparable leaf of a control plant not comprising the samemutation or transgene.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and capable of producing a leaf comprising a comparableleaf yield relative to a comparable leaf of a control plant notcomprising the same mutation or transgene. In one aspect, a comparableleaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1%of the level in a comparable leaf of a control plant not comprising thesame mutation or transgene. In an aspect, a comparable leaf yield isbetween 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3%and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%,between 12% and 13%, between 13% and 14%, between 14% and 15%, between15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and19%, or between 19% and 20% of the level in a comparable leaf of acontrol plant not comprising the same mutation or transgene. In afurther aspect, a comparable leaf yield is between 0.5% and 5%, between5% and 10%, or between 10% and 20% of the level in a comparable leaf ofa control plant not comprising the same mutation or transgene.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a low nicotine or low alkaloid-conferring mutationor transgene and exhibiting a comparable insect herbivory susceptibilityrelative to a comparable leaf of a control plant not comprising the samemutation or transgene. In one aspect, a comparable insect herbivorysusceptibility is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or1% of the level in a comparable leaf of a control plant not comprisingthe same mutation or transgene. In an aspect, a comparable insectherbivory susceptibility is between 0.5% and 1%, between 1% and 2%,between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9%and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%,between 14% and 15%, between 15% and 16%, between 16% and 17%, between17% and 18%, between 18% and 19%, or between 19% and 20% of the level ina comparable leaf of a control plant not comprising the same mutation ortransgene. In a further aspect, a comparable insect herbivorysusceptibility is between 0.5% and 5%, between 5% and 10%, or between10% and 20% of the level in a comparable leaf of a control plant notcomprising the same mutation or transgene.

Insect herbivory susceptibility level can be assayed by methods known inthe art, for example, in an insect feeding assay. In short, a quarterinch layer of 0.7% agar in water is added to a 100 mm Petri dish andallowed to solidify. Leaf discs are cut from the petri dish lid, placedin the plates and pushed gently into the agar. Leaf discs are taken fromplants at the 4-5 leaf stage. Discs were taken from lamina only toexclude major midribs. A single disc is taken from each of the fourlargest leaves of the plant generating 4 replicates per plant. Fourplants are sampled for a total of 16 biological replicates test line. Asingle budworm at the second instar stage is added to the leaf andallowed to feed for 48 hours at ambient temperature. After 48 hours thebudworm larvae are weighed and final larval weights are recorded.

Unless specified otherwise, measurements of alkaloid, polyamine, ornicotine levels (or another leaf chemistry or property characterization)or leaf grade index values mentioned herein for a tobacco plant,variety, cultivar, or line can refer to average measurements, including,for example, depending on the context, an average of multiple leaves ofa single plant or an average measurement from a population of tobaccoplants from a single variety, cultivar, or line. In an aspect, thenicotine, alkaloid, or polyamine level (or another leaf chemistry orproperty characterization) of a tobacco plant is measured after toppingin a pooled leaf sample collected from leaf number 3, 4, and 5 aftertopping. In another aspect, the nicotine, alkaloid, or polyamine level(or another leaf chemistry or property characterization) of a tobaccoplant is measured after topping in a leaf having the highest level ofnicotine, alkaloid, or polyamine (or another leaf chemistry or propertycharacterization). In an aspect, the nicotine, alkaloid, or polyaminelevel of a tobacco plant is measured after topping in leaf number 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30. In another aspect, the nicotine,alkaloid, or polyamine level (or another leaf chemistry or propertycharacterization) of a tobacco plant is measured after topping in a poolof two or more leaves with consecutive leaf numbers selected from thegroup consisting of leaf number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and30. In another aspect, the nicotine, alkaloid, or polyamine level (oranother leaf chemistry or property characterization) of a tobacco plantis measured after topping in a leaf with a leaf number selected from thegroup consisting of between 1 and 5, between 6 and 10, between 11 and15, between 16 and 20, between 21 and 25, and between 26 and 30. Inanother aspect, the nicotine, alkaloid, or polyamine level (or anotherleaf chemistry or property characterization) of a tobacco plant ismeasured after topping in a pool of two or more leaves with leaf numbersselected from the group consisting of between 1 and 5, between 6 and 10,between 11 and 15, between 16 and 20, between 21 and 25, and between 26and 30. In another aspect, the nicotine, alkaloid, or polyamine level(or another leaf chemistry or property characterization) of a tobaccoplant is measured after topping in a pool of three or more leaves withleaf numbers selected from the group consisting of between 1 and 5,between 6 and 10, between 11 and 15, between 16 and 20, between 21 and25, and between 26 and 30.

Alkaloid levels can be assayed by methods known in the art, for exampleby quantification based on gas-liquid chromatography, high performanceliquid chromatography, radio-immunoassays, and enzyme-linkedimmunosorbent assays.

As used herein, leaf numbering is based on the leaf position on atobacco stalk with leaf number 1 being the oldest leaf (at the base)after topping and the highest leaf number assigned to the youngest leaf(at the tip).

A population of tobacco plants or a collection of tobacco leaves fordetermining an average measurement (e.g., alkaloid or nicotine level orleaf grading) can be of any size, for example, 5, 10, 15, 20, 25, 30,35, 40, or 50. Industry-accepted standard protocols are followed fordetermining average measurements or grad index values.

As used herein, “topping” refers to the removal of the stalk apex,including the SAM, flowers, and up to several adjacent leaves, when atobacco plant is near vegetative maturity and around the start ofreproductive growth. Typically, tobacco plants are topped in the buttonstage (soon after the flower begins to appear). For example, greenhouseor field-grown tobacco plants can be topped when 50% of the plants haveat least one open flower. Topping a tobacco plant results in the loss ofapical dominance and also induce increased alkaloid production.

Typically, the nicotine, alkaloid, or polyamine level (or another leafchemistry or property characterization) of a tobacco plant is measuredabout 2 weeks after topping. Other time points can also be used. In anaspect, the nicotine, alkaloid, or polyamine level (or another leafchemistry or property characterization) of a tobacco plant is measuredabout 1, 2, 3, 4, or 5 weeks after topping. In another aspect, thenicotine, alkaloid, or polyamine level (or another leaf chemistry orproperty characterization) of a tobacco plant is measured about 3, 5, 7,10, 12, 14, 17, 19, or 21 days after topping.

As used herein, “similar growth conditions” refer to similarenvironmental conditions and/or agronomic practices for growing andmaking meaningful comparisons between two or more plant genotypes sothat neither environmental conditions nor agronomic practices wouldcontribute to or explain any difference observed between the two or moreplant genotypes. Environmental conditions include, for example, light,temperature, water (humidity), and nutrition (e.g., nitrogen andphosphorus). Agronomic practices include, for example, seeding,clipping, undercutting, transplanting, topping, and suckering. SeeChapters 4B and 4C of Tobacco, Production, Chemistry and Technology,Davis & Nielsen, eds., Blackwell Publishing, Oxford (1999), pp 70-103.

As used herein, “comparable leaves” refer to leaves having similar size,shape, age, and/or stalk position.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising an inducible promoter operably linked to atranscribable DNA sequence encoding a non-coding RNA for suppression ofan ornithine decarboxylase (ODC) gene. In one aspect, an induciblepromoter is a topping-inducible promoter. In an aspect, an induciblepromoter is also a tissue-specific or tissue-preferred promoter. In oneaspect, a tissue-specific or tissue-preferred promoter is specific orpreferred for one or more tissues or organs selected from the groupconsisting of shoot, root, leaf, stem, flower, sucker, root tip,mesophyll cells, epidermal cells, and vasculature. In a further aspect,a topping inducible promoter comprises a promoter sequence from atobacco nicotine demethylase gene, for example, CYP82E4, CYP82E5, orCYP82E10.

Various types of promoters can be used here, which are classifiedaccording to a variety of criteria relating to the pattern of expressionof a coding sequence or gene (including a transgene) operably linked tothe promoter, such as constitutive, developmental, tissue-specific,tissue-preferred, inducible, etc. Promoters that initiate transcriptionin all or most tissues of the plant are referred to as “constitutive”promoters. Promoters that initiate transcription during certain periodsor stages of development are referred to as “developmental” promoters.Promoters whose expression is enhanced in certain tissues of the plantrelative to other plant tissues are referred to as “tissue-enhanced” or“tissue-preferred” promoters. Thus, a “tissue-preferred” promoter causesrelatively higher or preferential expression in a specific tissue(s) ofthe plant, but with lower levels of expression in other tissue(s) of theplant. Promoters that express within a specific tissue(s) of the plant,with little or no expression in other plant tissues, are referred to as“tissue-specific” promoters. A promoter that expresses in a certain celltype of the plant is referred to as a “cell type specific” promoter. An“inducible” promoter is a promoter that initiates transcription inresponse to an environmental stimulus such as cold, drought, heat orlight, or other stimuli, such as wounding or chemical application. Apromoter may also be classified in terms of its origin, such as beingheterologous, homologous, chimeric, synthetic, etc. A “heterologous”promoter is a promoter sequence having a different origin relative toits associated transcribable sequence, coding sequence, or gene (ortransgene), and/or not naturally occurring in the plant species to betransformed. The term “heterologous” more broadly includes a combinationof two or more DNA molecules or sequences when such a combination is notnormally found in nature. For example, two or more DNA molecules orsequences would be heterologous with respect to each other if they arenormally found in different genomes or at different loci in the samegenome, or if they are not identically combined in nature.

In an aspect, an inducible promoter provides root specific or preferredexpression. In one aspect, a root specific or preferred induciblepromoter comprises a sequence selected from the group consisting of SEQID Nos: 1-11 and a functional fragment thereof. Table 1 provides acomparison of estimated leaf versus root specific expression leveldriven by SEQ ID Nos: 1-11.

In an aspect, an inducible promoter provides leaf specific or preferredexpression. In one aspect, a leaf specific or preferred induciblepromoter comprises a sequence selected from the group consisting of SEQID Nos: 12-21 and a functional fragment thereof. Table 2 provides acomparison of estimated leaf versus root specific expression leveldriven by SEQ ID Nos: 12-21.

TABLE 1 Exemplary inducible promoters for topping-responsive rootspecific or preferred expression Root Leaf 3 days after 3 days 4 wks 3days topping Gene Layby Before after after Layby Flowering after(nitrogen SEQ Id Id stage Topping Topping Topping Senescence stage timeTopping deficient) SEQ ID g78655 0.0 0.0 0.0 0.0 1.1 1.7 1.0 146.0 251.0NO: 1 SEQ ID g72021 0.0 0.0 1.7 0.7 3.3 4.3 6.9 501.7 468.7 NO: 2 SEQ IDg78252 3.2 0.0 0.0 1.5 0.0 0.9 3.0 77.3 167.8 NO: 3 SEQ ID g65720 0.00.0 1.7 0.0 3.8 0.9 0.0 33.5 6.0 NO: 4 SEQ ID g74108 3.2 3.3 0.0 0.7 3.62.6 4.0 81.6 142.9 NO: 5 SEQ ID g47466 12.7 31.6 0.0 69.6 43.2 0.0 1.022.3 10.6 NO: 6 SEQ ID g102868 3.2 3.3 0.0 0.0 0.2 0.9 1.0 36.9 23.4 NO:7 SEQ ID g23057 0.0 0.0 1.7 0.0 8.7 2.6 13.8 247.4 287.2 NO: 8 SEQ IDg34684 12.7 41.6 43.0 67.4 104.0 21.5 105.7 1947.5 2177.0 NO: 9 SEQ IDg105948 15.8 3.3 0.0 8.8 11.9 16.3 14.8 78.2 768.7 NO: 10 SEQ ID g81261145.6 68.3 72.3 90.2 73.1 44.7 115.6 2206.9 2112.0 NO: 11

TABLE 2 Exemplary inducible promoters for topping-responsive leafspecific or preferred expression 1 day 3 days 1 week 2 weeks 3 weeksBefore after after after after after Harvest SEQ Id Gene Topping ToppingTopping Topping Topping Topping time SEQ ID g2237 20 170 344 305 706 4321049 NO: 12 SEQ ID g31142 41 61 177 480 167 1530 892 NO: 13 SEQ IDg75488 41 9 306 534 47 2297 138 NO: 14 SEQ ID g94193 119 128 846 1650377 4337 1546 NO: 15 SEQ ID g34756 15 86 113 73 22 183 611 NO: 16 SEQ IDg104299 48 110 166 576 138 1521 994 NO: 17 SEQ ID g44810 21 19 99 80 492140 521 NO: 18 SEQ ID g71671 15 26 58 210 40 348 299 NO: 19 SEQ IDg29427 34 34 395 314 474 591 339 NO: 20 SEQ ID g49024 29 28 229 230 280382 595 NO: 21

In an aspect, an inducible promoter is a heterologous to the operablylinked transcribable DNA sequence. In one aspect, a transcribable DNAsequence encodes a non-coding RNA selected from the group consisting ofmicroRNA (miRNA), anti-sense RNA, small interfering RNA (siRNA), atrans-acting siRNA (ta-siRNA), and hairpin RNA (hpRNA). In an aspect, anon-coding RNA comprises a nucleotide sequence having at least 99.9%, atleast 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, atleast 95%, at least 94%, at least 93%, at least 92%, at least 91%, atleast 90%, at least 85%, at least 80%, or at least 75% identity to asequence selected from the group consisting of SEQ ID Nos: 35 and 36,and any portions thereof. In one aspect, a non-coding RNA is provided inan ODC RNAi construct comprising a nucleotide sequence having at least99%, at least 97%, at least 95%, at least 90%, at least 85%, at least80%, or at least 75% identity to SEQ ID No: 22.

“Alkaloids” are complex, nitrogen-containing compounds that naturallyoccur in plants, and have pharmacological effects in humans and animals.“Nicotine” is the primary natural alkaloid in commercialized cigarettetobacco and accounts for about 90 percent of the alkaloid content inNicotiana tabacum. Other major alkaloids in tobacco include cotinine,nornicotine, myosmine, nicotyrine, anabasine and anatabine. Minortobacco alkaloids include nicotine-n-oxide, N-methyl anatabine, N-methylanabasine, pseudooxynicotine, 2,3 dipyridyl and others.

In an aspect, tobacco plants provided herein comprise a lower level oftotal alkaloid or an individual alkaloid compared to a control tobaccoplant without a nic1 mutation and/or a nic2 mutation when grown insimilar growth conditions. In another aspect, tobacco plants providedherein comprise a lower level of one or more alkaloids selected from thegroup consisting of cotinine, nornicotine, myosmine, nicotyrine,anabasine and anatabine, compared to a control tobacco plant when grownin similar growth conditions. In an aspect, a lower alkaloid or nicotinelevel refers to an alkaloid or nicotine level of below 1%, below 2%,below 5%, below 8%, below 10%, below 12%, below 15%, below 20%, below25%, below 30%, below 40%, below 50%, below 60%, below 70%, or below 80%of the alkaloid or nicotine level of a control tobacco plant. In anotheraspect, a lower alkaloid or nicotine level refers to an alkaloid ornicotine level of about between 0.5% and 1%, between 1% and 2%, between2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%,between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and10%, between 11% and 12%, between 12% and 13%, between 13% and 14%,between 14% and 15%, between 15% and 16%, between 16% and 17%, between17% and 18%, between 18% and 19%, between 19% and 20%, between 21% and22%, between 22% and 23%, between 23% and 24%, between 24% and 25%,between 25% and 26%, between 26% and 27%, between 27% and 28%, between28% and 29%, or between 29% and 30% of the alkaloid or nicotine level ofa control tobacco plant. In a further aspect, a lower alkaloid ornicotine level refers to an alkaloid or nicotine level of about between0.5% and 5%, between 5% and 10%, between 10% and 20%, between 20% and30% of the alkaloid or nicotine level of a control tobacco plant.

Alkaloid levels can be assayed by methods known in the art, for exampleby quantification based on gas-liquid chromatography, high performanceliquid chromatography, radio-immunoassays, and enzyme-linkedimmunosorbent assays. For example, nicotinic alkaloid levels can bemeasured by a GC-FID method based on CORESTA Recommended Method No. 7,1987 and ISO Standards (ISO TC 126N 394 E. See also Hibi et al., PlantPhysiology 100: 826-35 (1992) for a method using gas-liquidchromatography equipped with a capillary column and an FID detector.Unless specified otherwise, all alkaloid levels described here aremeasured using a method in accordance with CORESTA Method No 62,Determination of Nicotine in Tobacco and Tobacco Products by GasChromatographic Analysis, February 2005, and those defined in theCenters for Disease Control and Prevention's Protocol for Analysis ofNicotine, Total Moisture and pH in Smokeless Tobacco Products, aspublished in the Federal Register Vol. 64, No. 55 Mar. 23, 1999 (and asamended in Vol. 74, No. 4, Jan. 7, 2009).

Alternatively, tobacco total alkaloids can be measured using asegmented-flow colorimetric method developed for analysis of tobaccosamples as adapted by Skalar Instrument Co (West Chester, Pa.) anddescribed by Collins et al., Tobacco Science 13:79-81 (1969). In short,samples of tobacco are dried, ground, and extracted prior to analysis oftotal alkaloids and reducing sugars. The method then employs an aceticacid/methanol/water extraction and charcoal for decolorization.Determination of total alkaloids was based on the reaction of cyanogenchloride with nicotine alkaloids in the presence of an aromatic amine toform a colored complex which is measured at 460 nm. Unless specifiedotherwise, total alkaloid levels or nicotine levels shown herein are ona dry weight basis (e.g., percent total alkaloid or percent nicotine).

In an aspect, tobacco plants provided herein comprise a lower level ofnicotine compared to a control tobacco plant without a nic1 mutationand/or a nic2 mutation when grown in similar growth conditions. In anaspect, a lower nicotine level refers to an average nicotine level ofbelow 1%, below 2%, below 5%, below 8%, below 10%, below 12%, below 15%,below 20%, below 25%, below 30%, below 40%, below 50%, below 60%, below70%, or below 80% of the average nicotine level of a control tobaccoplant. In another aspect, a lower nicotine level refers to an averagenicotine level of about between 0.5% and 1%, between 1% and 2%, between2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%,between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and10%, between 11% and 12%, between 12% and 13%, between 13% and 14%,between 14% and 15%, between 15% and 16%, between 16% and 17%, between17% and 18%, between 18% and 19%, between 19% and 20%, between 21% and22%, between 22% and 23%, between 23% and 24%, between 24% and 25%,between 25% and 26%, between 26% and 27%, between 27% and 28%, between28% and 29%, or between 29% and 30% of the average nicotine level of acontrol tobacco plant. In a further aspect, a lower nicotine levelrefers to an average nicotine level of about between 0.5% and 5%,between 5% and 10%, between 10% and 20%, between 20% and 30% of theaverage nicotine level of a control tobacco plant.

In an aspect, tobacco plants provided herein comprise an averagenicotine or total alkaloid level selected from the group consisting ofabout 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%,1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%,2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%,6%, 7%, 8%, and 9% on a dry weight basis. In another aspect, tobaccoplants provided herein comprise an average nicotine or total alkaloidlevel selected from the group consisting of about between 0.01% and0.02%, between 0.02% and 0.05%, between 0.05% and 0.75%, between 0.75%and 0.1%, between 0.1% and 0.15%, between 0.15% and 0.2%, between 0.2%and 0.3%, between 0.3% and 0.35%, between 0.35% and 0.4%, between 0.4%and 0.5%, between 0.5% and 0.6%, between 0.6% and 0.7%, between 0.7% and0.8%, between 0.8% and 0.9%, between 0.9% and 1%, between 1% and 1.1%,between 1.1% and 1.2%, between 1.2% and 1.3%, between 1.3% and 1.4%,between 1.4% and 1.5%, between 1.5% and 1.6%, between 1.6% and 1.7%,between 1.7% and 1.8%, between 1.8% and 1.9%, between 1.9% and 2%,between 2% and 2.1%, between 2.1% and 2.2%, between 2.2% and 2.3%,between 2.3% and 2.4%, between 2.4% and 2.5%, between 2.5% and 2.6%,between 2.6% and 2.7%, between 2.7% and 2.8%, between 2.8% and 2.9%,between 2.9% and 3%, between 3% and 3.1%, between 3.1% and 3.2%, between3.2% and 3.3%, between 3.3% and 3.4%, between 3.4% and 3.5%, and between3.5% and 3.6% on a dry weight basis. In a further aspect, tobacco plantsprovided herein comprise an average nicotine or total alkaloid levelselected from the group consisting of about between 0.01% and 0.1%,between 0.02% and 0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%,between 0.05% and 0.5%, between 0.75% and 1%, between 0.1% and 1.5%,between 0.15% and 2%, between 0.2% and 3%, and between 0.3% and 3.5% ona dry weight basis.

The present disclosure also provides tobacco plants having alterednicotine levels without negative impacts over other tobacco traits,e.g., leaf grade index value. In an aspect, a low-nicotine ornicotine-free tobacco variety provides cured tobacco of commerciallyacceptable grade. Tobacco grades are evaluated based on factorsincluding, but not limited to, the leaf stalk position, leaf size, leafcolor, leaf uniformity and integrity, ripeness, texture, elasticity,sheen (related to the intensity and the depth of coloration of the leafas well as the shine), hygroscopicity (the faculty of the tobacco leavesto absorb and to retain the ambient moisture), and green nuance or cast.Leaf grade can be determined, for example, using an Official StandardGrade published by the Agricultural Marketing Service of the USDepartment of Agriculture (7 U.S.C. § 511). See, e.g., Official StandardGrades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effectiveNov. 5, 1990 (55 F.R. 40645); Official Standard Grades for Flue-CuredTobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective Mar.27, 1989 (54 F.R. 7925); Official Standard Grades for PennsylvaniaSeedleaf Tobacco (U.S. Type 41), effective Jan. 8, 1965 (29 F.R. 16854);Official Standard Grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43,and 44), effective Dec. 8, 1963 (28 F.R. 11719 and 28 F.R. 11926);Official Standard Grades for Wisconsin Cigar-Binder Tobacco (U.S. Types54 and 55), effective Nov. 20, 1969 (34 F.R. 17061); Official StandardGrades for Wisconsin Cigar-Binder Tobacco (U.S. Types 54 and 55),effective Nov. 20, 1969 (34 F.R. 17061); Official Standard Grades forGeorgia and Florida Shade-Grown Cigar-Wrapper Tobacco (U.S. Type 62),Effective April 1971. A USDA grade index value can be determinedaccording to an industry accepted grade index. See, e.g., Bowman et al,Tobacco Science, 32:39-40(1988); Legacy Tobacco Document Library (BatesDocument #523267826-523267833, Jul. 1, 1988, Memorandum on the ProposedBurley Tobacco Grade Index); and Miller et al., 1990, Tobacco Intern.,192:55-57 (all foregoing references are incorporated by inference intheir entirety). In an aspect, a USDA grade index is a 0-100 numericalrepresentation of federal grade received and is a weighted average ofall stalk positions. A higher grade index indicates higher quality.Alternatively, leaf grade can be determined via hyper-spectral imaging.See e.g., WO 2011/027315 (published on Mar. 10, 2011, and incorporatedby inference in its entirety). A comparable leaf grade index indicates aleaf grade index that does not vary more than 30% above or below anappropriate control or comparator when comparing leaves from similarstalk positions. In an aspect, a comparable leaf grade index does notvary more than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%above or below an appropriate control or comparator when comparingleaves from similar stalk positions.

In an aspect, tobacco plants provided herein comprise a similar level ofone or more tobacco aroma compounds selected from the group consistingof 3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, acembrenoid, a sugar ester, and a reducing sugar, compared to controltobacco plants when grown in similar growth conditions. In anotheraspect, tobacco plants provided herein comprise a nic1 mutation, a nic2mutation, or a combination thereof having no impact over the level ofone or more tobacco aroma compounds selected from the group consistingof 3-methylvaleric acid, valeric acid, isovaleric acid, a labdenoid, acembrenoid, a sugar ester, and a reducing sugar.

As used herein, tobacco aroma compounds are compounds associated withthe flavor and aroma of tobacco smoke. These compounds include, but arenot limited to, 3-methylvaleric acid, valeric acid, isovaleric acid,cembrenoid and labdenoid diterpenes, and sugar esters. Concentrations oftobacco aroma compounds can be measured by any known metaboliteprofiling methods in the art including, without limitation, gaschromatography mass spectrometry (GC-MS), Nuclear Magnetic ResonanceSpectroscopy, liquid chromatography-linked mass spectrometry. See TheHandbook of Plant Metabolomics, edited by Weckwerth and Kahl,(Wiley-Blackwell) (May 28, 2013).

As used herein, “reducing sugar(s)” are any sugar (monosaccharide orpolysaccharide) that has a free or potentially free aldehdye or ketonegroup. Glucose and fructose act as nicotine buffers in cigarette smokeby reducing smoke pH and effectively reducing the amount of “free”unprotonated nicotine. Reducing sugars balances smoke flavor, forexample, by modifying the sensory impact of nicotine and other tobaccoalkaloids. An inverse relationship between sugar content and alkaloidcontent has been reported across tobacco varieties, within the samevariety, and within the same plant line caused by planting conditions.Reducing sugar levels can be measured using a segmented-flowcolorimetric method developed for analysis of tobacco samples as adaptedby Skalar Instrument Co (West Chester, Pa.) and described by Davis,Tobacco Science 20:139-144 (1976). For example, a sample is dialyzedagainst a sodium carbonate solution. Copper neocuproin is added to thesample and the solution is heated. The copper neocuproin chelate isreduced in the presence of sugars resulting in a colored complex whichis measured at 460 nm.

In an aspect, tobacco plants provided herein comprise one or morenon-naturally existing mutant alleles at nic1 and/or nic2 locus whichreduce or eliminate one or more gene activity from nic1 and/or nic2locus. In an aspect, these mutant alleles result in lower nicotinelevels. Mutant nic1 and/or nic2 alleles can be introduced by any methodknown in the art including random or targeted mutagenesis approaches.

Such mutagenesis methods include, without limitation, treatment of seedswith ethyl methylsulfate (EMS) (Hildering and Verkerk, In, The use ofinduced mutations in plant breeding. Pergamon press, pp 317-320, 1965)or UV-irradiation, X-rays, and fast neutron irradiation (see, forexample, Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; and Poehlman,Breeding Field Crops, Van Nostrand Reinhold, N.Y. (3.sup.rd ed), 1987),transposon tagging (Fedoroff et al., 1984; U.S. Pat. Nos. 4,732,856 and5,013,658), as well as T-DNA insertion methodologies (Hoekema et al.,1983; U.S. Pat. No. 5,149,645). EMS-induced mutagenesis consists ofchemically inducing random point mutations over the length of thegenome. Fast neutron mutagenesis consists of exposing seeds to neutronbombardment which causes large deletions through double stranded DNAbreakage. Transposon tagging comprises inserting a transposon within anendogenous gene to reduce or eliminate expression of the gene. The typesof mutations that may be present in a tobacco gene include, for example,point mutations, deletions, insertions, duplications, and inversions.Such mutations desirably are present in the coding region of a tobaccogene; however mutations in the promoter region, and intron, or anuntranslated region of a tobacco gene may also be desirable.

In addition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the present disclosure. See, McCallumet al. (2000) Nat. Biotechnol. 18:455-457. Mutations that impact geneexpression or that interfere with the function of genes can bedetermined using methods that are well known in the art. Insertionalmutations in gene exons usually result in null-mutants. Mutations inconserved residues can be particularly effective in inhibiting thefunction of a protein. In an aspect, tobacco plants comprise a nonsense(e.g., stop codon) mutation in one or more NCG genes described in U.S.Provisional Application Nos. 62/616,959 and 62/625,878, both of whichare incorporated by reference in their entirety.

In an aspect, the present disclosure also provides tobacco lines withaltered nicotine levels while maintaining commercially acceptable leafquality. These lines can be produced by introducing mutations into oneor more genes at nic1 and/or nic2 locus via precise genome engineeringtechnologies, for example, Transcription activator-like effectornucleases (TALENs), meganuclease, zinc finger nuclease, and a clusteredregularly-interspaced short palindromic repeats (CRISPR)/Cas9 system, aCRISPR/Cpf1 system, a CRISPR/Csm1 system, and a combination thereof(see, for example, U.S. Patent Application publication 2017/0233756).See, e.g., Gaj et al., Trends in Biotechnology, 31(7):397-405 (2013).

The screening and selection of mutagenized tobacco plants can be throughany methodologies known to those having ordinary skill in the art.Examples of screening and selection methodologies include, but are notlimited to, Southern analysis, PCR amplification for detection of apolynucleotide, Northern blots, RNase protection, primer-extension,RT-PCR amplification for detecting RNA transcripts, Sanger sequencing,Next Generation sequencing technologies (e.g., Illumina, PacBio, IonTorrent, 454), enzymatic assays for detecting enzyme or ribozymeactivity of polypeptides and polynucleotides, and protein gelelectrophoresis, Western blots, immunoprecipitation, and enzyme-linkedimmunoassays to detect polypeptides. Other techniques such as in situhybridization, enzyme staining, and immunostaining also can be used todetect the presence or expression of polypeptides and/orpolynucleotides. Methods for performing all of the referenced techniquesare known.

In an aspect, a tobacco plant or plant genome provided herein is mutatedor edited by a nuclease selected from the group consisting of ameganuclease, a zinc-finger nuclease (ZFN), a transcriptionactivator-like effector nuclease (TALEN), a CRISPR/Cas9 nuclease, aCRISPR/Cpf1 nuclease, or a CRISPR/Csm1 nuclease.

In an aspect, tobacco plants provided herein comprising a nic1 mutation,a nic2 mutation, or both, further comprises a transgene or mutationproviding an early-senescence trait. In one aspect, a mutation providingan early-senescence trait is yellow burley1 (−yb1). In an aspect, amutation providing an early-senescence trait is yellow burley2 (−yb2).In one aspect, a mutation providing an early-senescence trait is paleyellow (PY).

In an aspect, a tobacco plant, or part thereof, comprises relative to acontrol tobacco plant: a first genome modification providing a lowerlevel of nicotine or total alkaloid, and a second genome modificationproviding a comparable level of one or more traits selected from thegroup consisting of total leaf polyamine level, total root polyaminelevel, total leaf chlorophyll level, mesophyll cell number per leaf areaunit, and leaf epidermal cell size; and where the control plant does nothave both the first and the second genome modifications. In one aspect,a tobacco plant, or part thereof, comprises relative to a controltobacco plant: a first genome modification providing a lower level ofnicotine or total alkaloid, and a second genome modification providing acomparable level of total leaf polyamine level, where the control plantdoes not have both the first and the second genome modifications. In anaspect, a tobacco plant, or part thereof, comprises relative to acontrol tobacco plant: a first genome modification providing a lowerlevel of nicotine or total alkaloid, and a second genome modificationproviding a comparable level of total root polyamine level, where thecontrol plant does not have both the first and the second genomemodifications. In one aspect, a tobacco plant, or part thereof,comprises relative to a control tobacco plant: a first genomemodification providing a lower level of nicotine or total alkaloid, anda second genome modification providing a comparable level of total leafchlorophyll level, where the control plant does not have both the firstand the second genome modifications. In an aspect, a tobacco plant, orpart thereof, comprises relative to a control tobacco plant: a firstgenome modification providing a lower level of nicotine or totalalkaloid, and a second genome modification providing a comparable levelof mesophyll cell number per leaf area unit, where the control plantdoes not have both the first and the second genome modifications. In oneaspect, a tobacco plant, or part thereof, comprises relative to acontrol tobacco plant: a first genome modification providing a lowerlevel of nicotine or total alkaloid, and a second genome modificationproviding a comparable level of leaf epidermal cell size, where thecontrol plant does not have both the first and the second genomemodifications.

In an aspect, a first genome modification, a second genome modification,or both comprise a transgene, a mutation, or both. In one aspect, agenome modification, a second genome modification, or both comprise atransgene. In an aspect, a first genome modification, a second genomemodification, or both comprise a mutation. In one aspect, a first genomemodification, a second genome modification, or both are nottransgene-based. In an aspect, a first genome modification, a secondgenome modification, or both are not mutation-based.

In an aspect, tobacco plants provided herein comprise a first genomemodification providing a lower level of nicotine compared to a controltobacco plant. In one aspect, tobacco plants provided herein comprise afirst genome modification comprising a nic1 mutation, a nic2 mutation,or both. In an aspect, tobacco plants provided herein comprise atransgene targeting the Nic1 locus, a transgene targeting the Nic2locus, or both.

In an aspect, tobacco plants provided herein comprise a first genomemodification comprising a mutation in a gene or locus encoding a proteinselected from the group consisting of aspartate oxidase, agmatinedeiminase (AIC), arginase, diamine oxidase, arginine decarboxylase(ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithinedecarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622,NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF)transcription factor, nicotine uptake permease (NUP), and MATEtransporter. In one aspect, tobacco plants provided herein comprise afirst genome modification comprises a transgene targeting andsuppressing a gene or locus encoding a protein selected from the groupconsisting of aspartate oxidase, agmatine deiminase (AIC), arginase,diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase(MPO), NADH dehydrogenase, ornithine decarboxylase (ODC),phosphoribosylanthranilate isomerase (PRAI), putrescineN-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT),and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2,Nic1, Nic2, ethylene response factor (ERF) transcription factor,nicotine uptake permease (NUP), and MATE transporter.

In an aspect, tobacco plants provided herein comprise a first genomemodification comprising a mutation in a gene or locus encoding a proteinselected from the group consisting of ERF32, ERF34, ERF39, ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168. In one aspect,tobacco plants provided herein comprise a first genome modificationcomprises a transgene targeting and suppressing a gene or locus encodinga protein selected from the group consisting of ERF32, ERF34, ERF39,ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

In an aspect, a tobacco plant or plant genome provided is mutated oredited to have one or more mutations in one or more NCG genes selectedfrom the group consisting of NCG1 to NCG35, where the one or moremutations reduce or eliminate the activity or expression of the one ormore NCG genes. In an aspect, a tobacco plant or plant genome providedis mutated or edited to have one or more mutations in one or more NCGgenes selected from the group consisting of NCG1, NCG2, NCG11, NCG12,NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30,and NCG35, where the one or more mutations reduce or eliminate theactivity or expression of the one or more NCG genes. In an aspect, atobacco plant or plant genome provided is mutated or edited to have oneor more mutations in one or more NCG genes selected from the groupconsisting of NCG1 to NCG21, where the one or more mutations reduce oreliminate the activity or expression of the one or more NCG genes. In anaspect, a tobacco plant or plant genome provided is mutated or edited tohave one or more mutations in one or more NCG genes selected from thegroup consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17,where the one or more mutations reduce or eliminate the activity orexpression of the one or more NCG genes. In an aspect, a tobacco plantor plant genome provided is mutated or edited to have one or moremutations in one or more NCG genes selected from the group consisting ofNCG2, NCG12, NCG15, NCG16, and NCG17, where the one or more mutationsreduce or eliminate the activity or expression of the one or more NCGgenes. In an aspect, a tobacco plant or plant genome provided is mutatedor edited to have one or more mutations in one or more NCG genesselected from the group consisting of NCG12, NCG15, NCG16, and NCG17,where the one or more mutations reduce or eliminate the activity orexpression of the one or more NCG genes. In another aspect, an edited ormutated tobacco plant having one or more, two or more, three or more,four or more, or five or more NCG mutations further comprises one ormore mutations in one or more, two or more, three or more, four or more,or five or more ERF genes selected from the group consisting of ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the one or moremutations reduce or eliminate the activity or expression of the one ormore ERF genes.

In an aspect, a tobacco plant or plant genome provided is mutated oredited to have two or more mutations in two or more NCG genes selectedfrom the group consisting of NCG1 to NCG35, where the two or moremutations reduce or eliminate the activity or expression of the two ormore NCG genes. In an aspect, a tobacco plant or plant genome providedis mutated or edited to have two or more mutations in two or more NCGgenes selected from the group consisting of NCG1, NCG2, NCG11, NCG12,NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30,and NCG35, where the two or more mutations reduce or eliminate theactivity or expression of the two or more NCG genes. In an aspect, atobacco plant or plant genome provided is mutated or edited to have twoor more mutations in two or more NCG genes selected from the groupconsisting of NCG1 to NCG21, where the two or more mutations reduce oreliminate the activity or expression of the one or more NCG genes. In anaspect, a tobacco plant or plant genome provided is mutated or edited tohave two or more mutations in two or more NCG genes selected from thegroup consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17,where the two or more mutations reduce or eliminate the activity orexpression of the one or more NCG genes. In an aspect, a tobacco plantor plant genome provided is mutated or edited to have two or moremutations in two or more NCG genes selected from the group consisting ofNCG2, NCG12, NCG15, NCG16, and NCG17, where the two or more mutationsreduce or eliminate the activity or expression of the two or more NCGgenes. In an aspect, a tobacco plant or plant genome provided is mutatedor edited to have two or more mutations in two or more NCG genesselected from the group consisting of NCG12, NCG15, NCG16, and NCG17,where the two or more mutations reduce or eliminate the activity orexpression of the two or more NCG genes. In another aspect, an edited ormutated tobacco plant having two or more NCG mutations further comprisestwo or more mutations in two or more ERF genes selected from the groupconsisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168,where the two or more mutations reduce or eliminate the activity orexpression of the two or more ERF genes.

In an aspect, a tobacco plant or plant genome provided is mutated oredited to have three or more mutations in three or more NCG genesselected from the group consisting of NCG1 to NCG35, where the three ormore mutations reduce or eliminate the activity or expression of thethree or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided is mutated or edited to have three or more mutations in threeor more NCG genes selected from the group consisting of NCG1, NCG2,NCG11, NCG12, NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26,NCG29, NCG30, and NCG35, where the three or more mutations reduce oreliminate the activity or expression of the three or more NCG genes. Inan aspect, a tobacco plant or plant genome provided is mutated or editedto have three or more mutations in three or more NCG genes selected fromthe group consisting of NCG1 to NCG21, where the three or more mutationsreduce or eliminate the activity or expression of the three or more NCGgenes. In an aspect, a tobacco plant or plant genome provided is mutatedor edited to have three or more mutations in three or more NCG genesselected from the group consisting of NCG1, NCG2, NCG11, NCG12, NCG15,NCG16, and NCG17, where the three or more mutations reduce or eliminatethe activity or expression of the three or more NCG genes. In an aspect,a tobacco plant or plant genome provided is mutated or edited to havethree or more mutations in three or more NCG genes selected from thegroup consisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where thethree or more mutations reduce or eliminate the activity or expressionof the three or more NCG genes. In an aspect, a tobacco plant or plantgenome provided is mutated or edited to have three or more mutations inthree or more NCG genes selected from the group consisting of NCG12,NCG15, NCG16, and NCG17, where the three or more mutations reduce oreliminate the activity or expression of the three or more NCG genes. Inanother aspect, an edited or mutated tobacco plant having three or moreNCG mutations further comprises three or more mutations in three or moreERF genes selected from the group consisting of ERF189, ERF115, ERF221,ERF104, ERF179, ERF17, and ERF168, where the three or more mutationsreduce or eliminate the activity or expression of the three or more ERFgenes.

In an aspect, a tobacco plant or plant genome provided is mutated oredited to have four or more mutations in four or more NCG genes selectedfrom the group consisting of NCG1 to NCG35, where the four or moremutations reduce or eliminate the activity or expression of the four ormore NCG genes. In an aspect, a tobacco plant or plant genome providedis mutated or edited to have four or more mutations in four or more NCGgenes selected from the group consisting of NCG1, NCG2, NCG11, NCG12,NCG13, NCG15, NCG16, NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30,and NCG35, where the four or more mutations reduce or eliminate theactivity or expression of the four or more NCG genes. In an aspect, atobacco plant or plant genome provided is mutated or edited to have fouror more mutations in four or more NCG genes selected from the groupconsisting of NCG1 to NCG21, where the four or more mutations reduce oreliminate the activity or expression of the four or more NCG genes. Inan aspect, a tobacco plant or plant genome provided is mutated or editedto have four or more mutations in four or more NCG genes selected fromthe group consisting of NCG1, NCG2, NCG11, NCG12, NCG15, NCG16, andNCG17, where the four or more mutations reduce or eliminate the activityor expression of the four or more NCG genes. In an aspect, a tobaccoplant or plant genome provided is mutated or edited to have four or moremutations in four or more NCG genes selected from the group consistingof NCG2, NCG12, NCG15, NCG16, and NCG17, where the four or moremutations reduce or eliminate the activity or expression of the four ormore NCG genes. In an aspect, a tobacco plant or plant genome providedis mutated or edited to have four or more mutations in four or more NCGgenes selected from the group consisting of NCG12, NCG15, NCG16, andNCG17, where the four or more mutations reduce or eliminate the activityor expression of the four or more NCG genes. In another aspect, anedited or mutated tobacco plant having four or more NCG mutationsfurther comprises four or more mutations in four or more ERF genesselected from the group consisting of ERF189, ERF115, ERF221, ERF104,ERF179, ERF17, and ERF168, where the four or more mutations reduce oreliminate the activity or expression of the four or more ERF genes.

In an aspect, a tobacco plant or plant genome provided comprises one ormore transgenes targeting one or more NCG genes selected from the groupconsisting of NCG1 to NCG35, where the one or more transgenes reduce oreliminate the activity or expression of the one or more NCG genes. In anaspect, a tobacco plant or plant genome provided comprises one or moretransgenes targeting one or more NCG genes selected from the groupconsisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17,NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the one ormore transgenes reduce or eliminate the activity or expression of theone or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises one or more transgenes targeting one or more NCGgenes selected from the group consisting of NCG1 to NCG21, where the oneor more transgenes reduce or eliminate the activity or expression of theone or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises one or more transgenes targeting one or more NCGgenes selected from the group consisting of NCG1, NCG2, NCG11, NCG12,NCG15, NCG16, and NCG17, where the one or more transgenes reduce oreliminate the activity or expression of the one or more NCG genes. In anaspect, a tobacco plant or plant genome provided comprises one or moretransgenes targeting one or more NCG genes selected from the groupconsisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the one ormore transgenes reduce or eliminate the activity or expression of theone or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises one or more transgenes targeting one or more NCGgenes selected from the group consisting of NCG12, NCG15, NCG16, andNCG17, where the one or more transgenes reduce or eliminate the activityor expression of the one or more NCG genes. In another aspect, a tobaccoplant having one or more, two or more, three or more, four or more, orfive or more NCG-targeting transgenes further comprises one or moremutations in one or more, two or more, three or more, four or more, orfive or more ERF genes selected from the group consisting of ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the one or moremutations reduce or eliminate the activity or expression of the one ormore ERF genes. In another aspect, a tobacco plant having one or more,two or more, three or more, four or more, or five or more NCG-targetingtransgenes further comprises one or more transgenes targeting one ormore, two or more, three or more, four or more, or five or more ERFgenes selected from the group consisting of ERF189, ERF115, ERF221,ERF104, ERF179, ERF17, and ERF168, where the one or more ERF-targetingtransgenes reduce or eliminate the activity or expression of the one ormore ERF genes.

In an aspect, a tobacco plant or plant genome provided comprises two ormore transgenes targeting two or more NCG genes selected from the groupconsisting of NCG1 to NCG35, where the two or more transgenes reduce oreliminate the activity or expression of the two or more NCG genes. In anaspect, a tobacco plant or plant genome provided comprises two or moretransgenes targeting two or more NCG genes selected from the groupconsisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17,NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the two ormore transgenes reduce or eliminate the activity or expression of thetwo or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises two or more transgenes targeting two or more NCGgenes selected from the group consisting of NCG1 to NCG21, where the twoor more transgenes reduce or eliminate the activity or expression of theone or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises two or more transgenes targeting two or more NCGgenes selected from the group consisting of NCG1, NCG2, NCG11, NCG12,NCG15, NCG16, and NCG17, where the two or more transgenes reduce oreliminate the activity or expression of the one or more NCG genes. In anaspect, a tobacco plant or plant genome provided comprises two or moretransgenes targeting two or more NCG genes selected from the groupconsisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the two ormore transgenes reduce or eliminate the activity or expression of thetwo or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises two or more transgenes targeting two or more NCGgenes selected from the group consisting of NCG12, NCG15, NCG16, andNCG17, where the two or more transgenes reduce or eliminate the activityor expression of the two or more NCG genes. In another aspect, a tobaccoplant having two or more NCG-targeting transgenes further comprises twoor more mutations in two or more ERF genes selected from the groupconsisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168,where the two or more mutations reduce or eliminate the activity orexpression of the two or more ERF genes. In another aspect, a tobaccoplant having two or more NCG-targeting transgenes further comprises twoor more transgenes targeting two or more ERF genes selected from thegroup consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, andERF168, where the two or more ERF-targeting transgenes reduce oreliminate the activity or expression of the two or more ERF genes.

In an aspect, a tobacco plant or plant genome provided comprises threeor more transgenes targeting three or more NCG genes selected from thegroup consisting of NCG1 to NCG35, where the three or more transgenesreduce or eliminate the activity or expression of the three or more NCGgenes. In an aspect, a tobacco plant or plant genome provided comprisesthree or more transgenes targeting three or more NCG genes selected fromthe group consisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16,NCG17, NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where thethree or more transgenes reduce or eliminate the activity or expressionof the three or more NCG genes. In an aspect, a tobacco plant or plantgenome provided comprises three or more transgenes targeting three ormore NCG genes selected from the group consisting of NCG1 to NCG21,where the three or more transgenes reduce or eliminate the activity orexpression of the three or more NCG genes. In an aspect, a tobacco plantor plant genome provided comprises three or more transgenes targetingthree or more NCG genes selected from the group consisting of NCG1,NCG2, NCG11, NCG12, NCG15, NCG16, and NCG17, where the three or moretransgenes reduce or eliminate the activity or expression of the threeor more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises three or more transgenes targeting three or more NCGgenes selected from the group consisting of NCG2, NCG12, NCG15, NCG16,and NCG17, where the three or more transgenes reduce or eliminate theactivity or expression of the three or more NCG genes. In an aspect, atobacco plant or plant genome provided comprises three or moretransgenes targeting three or more NCG genes selected from the groupconsisting of NCG12, NCG15, NCG16, and NCG17, where the three or moretransgenes reduce or eliminate the activity or expression of the threeor more NCG genes. In another aspect, a tobacco plant having three ormore NCG-targeting transgenes further comprises three or more mutationsin three or more ERF genes selected from the group consisting of ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168, where the three ormore mutations reduce or eliminate the activity or expression of thethree or more ERF genes. In another aspect, a tobacco plant having threeor more NCG-targeting transgenes further comprises three or moretransgenes targeting three or more ERF genes selected from the groupconsisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168,where the three or more ERF-targeting transgenes reduce or eliminate theactivity or expression of the three or more ERF genes.

In an aspect, a tobacco plant or plant genome provided comprises four ormore transgenes targeting four or more NCG genes selected from the groupconsisting of NCG1 to NCG35, where the four or more transgenes reduce oreliminate the activity or expression of the four or more NCG genes. Inan aspect, a tobacco plant or plant genome provided comprises four ormore transgenes targeting four or more NCG genes selected from the groupconsisting of NCG1, NCG2, NCG11, NCG12, NCG13, NCG15, NCG16, NCG17,NCG21, NCG22, NCG24, NCG26, NCG29, NCG30, and NCG35, where the four ormore transgenes reduce or eliminate the activity or expression of thefour or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises four or more transgenes targeting four or more NCGgenes selected from the group consisting of NCG1 to NCG21, where thefour or more transgenes reduce or eliminate the activity or expressionof the four or more NCG genes. In an aspect, a tobacco plant or plantgenome provided comprises four or more transgenes targeting four or moreNCG genes selected from the group consisting of NCG1, NCG2, NCG11,NCG12, NCG15, NCG16, and NCG17, where the four or more transgenes reduceor eliminate the activity or expression of the four or more NCG genes.In an aspect, a tobacco plant or plant genome provided comprises four ormore transgenes targeting four or more NCG genes selected from the groupconsisting of NCG2, NCG12, NCG15, NCG16, and NCG17, where the four ormore transgenes reduce or eliminate the activity or expression of thefour or more NCG genes. In an aspect, a tobacco plant or plant genomeprovided comprises four or more transgenes targeting four or more NCGgenes selected from the group consisting of NCG12, NCG15, NCG16, andNCG17, where the four or more transgenes reduce or eliminate theactivity or expression of the four or more NCG genes. In another aspect,a tobacco plant having four or more NCG-targeting transgenes furthercomprises four or more mutations in four or more ERF genes selected fromthe group consisting of ERF189, ERF115, ERF221, ERF104, ERF179, ERF17,and ERF168, where the four or more mutations reduce or eliminate theactivity or expression of the four or more ERF genes. In another aspect,a tobacco plant having four or more NCG-targeting transgenes furthercomprises four or more transgenes targeting four or more ERF genesselected from the group consisting of ERF189, ERF115, ERF221, ERF104,ERF179, ERF17, and ERF168, where the four or more ERF-targetingtransgenes reduce or eliminate the activity or expression of the four ormore ERF genes.

In an aspect, tobacco plants provided herein comprise second genomemodification comprising an inducible promoter operably linked to atranscribable DNA sequence encoding a non-coding RNA for suppression ofan ornithine decarboxylase (ODC) gene. In another aspect, a tobaccoplant is provided having suppressed MYB8 activity via either transgenesuppression, mutagenesis, or targeted genome editing. For simplicity,every instance here mentioning ODC suppression (e.g., operably linked toany particular type of promoters) is equally applicable to MYB8suppression.

In an aspect, tobacco plants provided herein comprise a reduced amountof total conjugated polyamines in leaves relative to the control tobaccoplant. In one aspect, tobacco plants provided herein comprise a reducedamount of total conjugated polyamines in roots relative to the controltobacco plant. Used here, conjugated polyamines include, but are notlimited to, soluble conjugated polyamines such as phenolamidescontaining a backbone consisting of a free polyamine (e.g., putrescine,spermine, and/or spermidine) conjugated with one or morephenylpropanoids such as ferulic, caffeic and courmaric acids.Conjugated polyamines also include, but are not limited to, insolubleconjugated polyamines incorporated into structural polymers such aslignin. In an aspect, tobacco plants provided herein comprise a reducedamount of total free polyamines (e.g., putrescine, spermine, andspermidine) in leaves relative to the control tobacco plant. In oneaspect, tobacco plants provided herein comprise a reduced amount oftotal conjugated polyamines in roots relative to the control tobaccoplant. In an aspect, tobacco plants provided herein comprise a reducedamount of total conjugated form of one or more polyamines selected fromthe group consisting of putrescine, spermidine and spermine in leavesrelative to the control tobacco plant. In one aspect, tobacco plantsprovided herein comprise a reduced amount of total conjugated form ofone or more polyamines selected from the group consisting of putrescine,spermidine and spermine in roots relative to the control tobacco plant.In an aspect, tobacco plants provided herein comprise a reduced amountof total free form of one or more polyamines selected from the groupconsisting of putrescine, spermidine and spermine in leaves relative tothe control tobacco plant. In one aspect, tobacco plants provided hereincomprise a reduced amount of total conjugated form of one or morepolyamines selected from the group consisting of putrescine, spermidineand spermine in roots relative to the control tobacco plant.

In an aspect, a characteristic or a trait of a tobacco plant describedhere are measured at a time selected from the group consisting ofimmediately before flowering, at topping, 1 week-post-topping (WPT), 2WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest. In oneaspect, tobacco plants provided herein comprising a first and a secondgenome modification are capable of producing a leaf with a leaf gradecomparable to that of a leaf from a control plant. In an aspect, tobaccoplants provided herein comprising a first and a second genomemodification have a total leaf yield comparable to a control plant.

As used herein, “editing” or “genome editing” refers to targetedmutagenesis of at least 1, at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, or at least 10nucleotides of an endogenous plant genome nucleic acid sequence, orremoval or replacement of an endogenous plant genome nucleic acidsequence. In an aspect, an edited nucleic acid sequence provided has atleast 99.9%, at least 99.5%, at least 99%, at least 98%, at least 97%,at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, atleast 91%, at least 90%, at least 85%, at least 80%, or at least 75%sequence identity with an endogenous nucleic acid sequence. In anaspect, an edited nucleic acid sequence provided has at least 99.9%, atleast 99.5%, at least 99%, at least 98%, at least 97%, at least 96%, atleast 95%, at least 94%, at least 93%, at least 92%, at least 91%, atleast 90%, at least 85%, at least 80%, or at least 75% sequence identitywith SEQ ID Nos: 23-28, and fragments thereof. In another aspect, anedited nucleic acid sequence provided has at least 99.9%, at least99.5%, at least 99%, at least 98%, at least 97%, at least 96%, at least95%, at least 94%, at least 93%, at least 92%, at least 91%, at least90%, at least 85%, at least 80%, or at least 75% sequence identity witha polynucleotide encoding a polypeptide selected from the groupconsisting of SEQ ID NOs:29-34.

Meganucleases, ZFNs, TALENs, CRISPR/Cas9, CRISPR/Csm1 and CRISPR/Cpf1induce a double-strand DNA break at a target site of a genomic sequencethat is then repaired by the natural processes of homologousrecombination (HR) or non-homologous end-joining (NHEJ). Sequencemodifications then occur at the cleaved sites, which can includedeletions or insertions that result in gene disruption in the case ofNHEJ, or integration of donor nucleic acid sequences by HR. In anaspect, a method provided comprises editing a plant genome with anuclease provided to mutate at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, or more than 10 nucleotides in the plant genome via HR with a donorpolynucleotide. In an aspect, a mutation provided is caused by genomeediting using a nuclease. In another aspect, a mutation provided iscaused by non-homologous end-joining or homologous recombination.

In an aspect, a mutation provided here provides a dominant mutant thatactivates the expression or activity of a gene of interest, e.g., a geneselected from the group consisting of a biosynthetic enzyme, aregulatory transcription factor, a transporter, a catabolic enzyme, or acombination thereof, for one or more antioxidants.

Meganucleases, which are commonly identified in microbes, are uniqueenzymes with high activity and long recognition sequences (>14 bp)resulting in site-specific digestion of target DNA. Engineered versionsof naturally occurring meganucleases typically have extended DNArecognition sequences (for example, 14 to 40 bp). The engineering ofmeganucleases can be more challenging than that of ZFNs and TALENsbecause the DNA recognition and cleavage functions of meganucleases areintertwined in a single domain. Specialized methods of mutagenesis andhigh-throughput screening have been used to create novel meganucleasevariants that recognize unique sequences and possess improved nucleaseactivity.

ZFNs are synthetic proteins consisting of an engineered zinc fingerDNA-binding domain fused to the cleavage domain of the FokI restrictionendonuclease. ZFNs can be designed to cleave almost any long stretch ofdouble-stranded DNA for modification of the zinc finger DNA-bindingdomain. ZFNs form dimers from monomers composed of a non-specific DNAcleavage domain of FokI endonuclease fused to a zinc finger arrayengineered to bind a target DNA sequence.

The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-fingerarrays. The amino acids at positions −1, +2, +3, and +6 relative to thestart of the zinc finger ∞-helix, which contribute to site-specificbinding to the target DNA, can be changed and customized to fit specifictarget sequences. The other amino acids form the consensus backbone togenerate ZFNs with different sequence specificities. Rules for selectingtarget sequences for ZFNs are known in the art.

The FokI nuclease domain requires dimerization to cleave DNA andtherefore two ZFNs with their C-terminal regions are needed to bindopposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFNmonomer can cute the target site if the two-ZF-binding sites arepalindromic. The term ZFN, as used herein, is broad and includes amonomeric ZFN that can cleave double stranded DNA without assistancefrom another ZFN. The term ZFN is also used to refer to one or bothmembers of a pair of ZFNs that are engineered to work together to cleaveDNA at the same site.

Without being limited by any scientific theory, because the DNA-bindingspecificities of zinc finger domains can in principle be re-engineeredusing one of various methods, customized ZFNs can theoretically beconstructed to target nearly any gene sequence. Publicly availablemethods for engineering zinc finger domains include Context-dependentAssembly (CoDA), Oligomerized Pool Engineering (OPEN), and ModularAssembly.

TALENs are artificial restriction enzymes generated by fusing thetranscription activator-like effector (TALE) DNA binding domain to aFokI nuclease domain. When each member of a TALEN pair binds to the DNAsites flanking a target site, the FokI monomers dimerize and cause adouble-stranded DNA break at the target site. The term TALEN, as usedherein, is broad and includes a monomeric TALEN that can cleave doublestranded DNA without assistance from another TALEN. The term TALEN isalso used to refer to one or both members of a pair of TALENs that worktogether to cleave DNA at the same site.

Transcription activator-like effectors (TALEs) can be engineered to bindpractically any DNA sequence. TALE proteins are DNA-binding domainsderived from various plant bacterial pathogens of the genus Xanthomonas.The Xanthomonas pathogens secrete TALEs into the host plant cell duringinfection. The TALE moves to the nucleus, where it recognizes and bindsto a specific DNA sequence in the promoter region of a specific DNAsequence in the promoter region of a specific gene in the host genome.TALE has a central DNA-binding domain composed of 13-28 repeat monomersof 33-34 amino acids. The amino acids of each monomer are highlyconserved, except for hypervariable amino acid residues at positions 12and 13. The two variable amino acids are called repeat-variablediresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDspreferentially recognize adenine, thymine, cytosine, andguanine/adenine, respectively, and modulation of RVDs can recognizeconsecutive DNA bases. This simple relationship between amino acidsequence and DNA recognition has allowed for the engineering of specificDNA binding domains by selecting a combination of repeat segmentscontaining the appropriate RVDs.

Besides the wild-type FokI cleavage domain, variants of the FokIcleavage domain with mutations have been designed to improve cleavagespecificity and cleavage activity. The FokI domain functions as a dimer,requiring two constructs with unique DNA binding domains for sites inthe target genome with proper orientation and spacing. Both the numberof amino acid residues between the TALEN DNA binding domain and the FokIcleavage domain and the number of bases between the two individual TALENbinding sites are parameters for achieving high levels of activity.

A relationship between amino acid sequence and DNA recognition of theTALE binding domain allows for designable proteins. Software programssuch as DNA Works can be used to design TALE constructs. Other methodsof designing TALE constructs are known to those of skill in the art. SeeDoyle et al, Nucleic Acids Research (2012) 40: W117-122; Cermak et al.,Nucleic Acids Research (2011). 39:e82; andtale-nt.cac.cornell.edu/about.

A CRISPR/Cas9 system, CRISPR/Csm1, or a CRISPR/Cpf1 system arealternatives to the FokI-based methods ZFN and TALEN. The CRISPR systemsare based on RNA-guided engineered nucleases that use complementary basepairing to recognize DNA sequences at target sites.

CRISPR/Cas9, CRISPR/Csm1, and a CRISPR/Cpf1 systems are part of theadaptive immune system of bacteria and archaea, protecting them againstinvading nucleic acids such as viruses by cleaving the foreign DNA in asequence-dependent manner. The immunity is acquired by the integrationof short fragments of the invading DNA known as spacers between twoadjacent repeats at the proximal end of a CRISPR locus. The CRISPRarrays, including the spacers, are transcribed during subsequentencounters with invasive DNA and are processed into small interferingCRISPR RNAs (crRNAs) approximately 40 nt in length, which combine withthe trans-activating CRISPR RNA (tracrRNA) to activate and guide theCas9 nuclease. This cleaves homologous double-stranded DNA sequencesknown as protospacers in the invading DNA. A prerequisite for cleavageis the presence of a conserved protospacer-adjacent motif (PAM)downstream of the target DNA, which usually has the sequence 5-NGG-3 butless frequently NAG. Specificity is provided by the so-called “seedsequence” approximately 12 bases upstream of the PAM, which must matchbetween the RNA and target DNA. Cpf1 and Csm1 act in a similar manner toCas9, but Cpf1 and Csm1 do not require a tracrRNA.

In still another aspect, a tobacco plant provided further comprises oneor more mutations in one or more loci encoding a nicotine demethylase(e.g., CYP82E4, CYP82E5, CYP82E10) that confer reduced amounts ofnornicotine (See U.S. Pat. Nos. 8,319,011; 8,124,851; 9,187,759;9,228,194; 9,228,195; 9,247,706) compared to control plant lacking oneor more mutations in one or more loci encoding a nicotine demethylase.In an aspect, a modified tobacco plant described further comprisesreduced nicotine demethylase activity compared to a control plant whengrown and cured under comparable conditions. In a further aspect, atobacco plant provided further comprises one or more mutations ortransgenes providing an elevated level of one or more antioxidants (SeeU.S. patent application Ser. No. 15/727,523 and PCT Application No.PCT/US2017/055618). In another aspect, a tobacco plant provided furthercomprises one or more mutations or transgenes providing a reduced levelof one or more TSNAs (such as N′-nitrosonornicotine (NNN),4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine(NAT) N′-nitrosoanabasine (NAB)).

The present disclosure also provides compositions and methods forinhibiting the expression or function of one or more genes involved inpolyamine biosynthesis or regulation thereof, in a plant, particularlyplants of the Nicotiana genus, including tobacco plants of the variouscommercial varieties.

In an aspect, the present disclosure provides tobacco plants, or partthereof, comprising a heterologous expression cassette comprising an ODCinhibitory sequence. In another aspect, tobacco plants, or part thereof,comprise a heterologous expression cassette comprising an inhibitorysequence of a gene comprising a sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%, or 100% identity to a sequence selected from the group consistingof SEQ ID NOs: 23-28, and fragments thereof, where the inhibitorysequence is operably linked to a promoter that is functional in a plantcell, and where the inhibitory sequence has at least 90% sequenceidentity to a fragment of at least 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nucleotides of the sequence having at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%identity to a sequence selected from the group consisting of SEQ ID NOs:23-28, and fragments thereof.

As used herein, the terms “inhibit,” “inhibition,” and “inhibiting” aredefined as any method known in the art or described herein thatdecreases the expression or function of a gene product of interest(e.g., a target gene product). “Inhibition” can be in the context of acomparison between two plants, for example, a genetically altered plantversus a wild-type plant. Alternatively, inhibition of expression orfunction of a target gene product can be in the context of a comparisonbetween plant cells, organelles, organs, tissues, or plant parts withinthe same plant or between different plants, and includes comparisonsbetween developmental or temporal stages within the same plant or plantpart or between plants or plant parts. “Inhibition” includes anyrelative decrement of function or production of a gene product ofinterest, up to and including complete elimination of function orproduction of that gene product. The term “inhibition” encompasses anymethod or composition that down-regulates translation and/ortranscription of the target gene product or functional activity of thetarget gene product. In an aspect, the mRNA or protein level of one ormore genes in a modified plant is less than 95%, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, less than 4%,less than 3%, less than 2%, or less than 1% of the mRNA or protein levelof the same gene in a plant that is not a mutant or that has not beengenetically modified to inhibit the expression of that gene.

The term “inhibitory sequence” encompasses any polynucleotide orpolypeptide sequence capable of inhibiting the expression or function ofa gene involved in nicotine biosynthesis regulation from Nic1b locus ina plant, such as full-length polynucleotide or polypeptide sequences,truncated polynucleotide or polypeptide sequences, fragments ofpolynucleotide or polypeptide sequences, variants of polynucleotide orpolypeptide sequences, sense-oriented nucleotide sequences,antisense-oriented nucleotide sequences, the complement of a sense- orantisense-oriented nucleotide sequence, inverted regions of nucleotidesequences, hairpins of nucleotide sequences, double-stranded nucleotidesequences, single-stranded nucleotide sequences, combinations thereof,and the like. The term “polynucleotide sequence” includes sequences ofRNA, DNA, chemically modified nucleic acids, nucleic acid analogs,combinations thereof, and the like.

Inhibitory sequences are designated by the name of the target geneproduct. Thus, a “ODC inhibitory sequence” refers to an inhibitorysequence that is capable of inhibiting the expression of an ODC geneinvolved in polyamine biosynthesis regulation in a plant, for example,at the level of transcription and/or translation, or which is capable ofinhibiting the function of a gene product. When the phrase “capable ofinhibiting” is used in the context of a polynucleotide inhibitorysequence, it is intended to mean that the inhibitory sequence itselfexerts the inhibitory effect; or, where the inhibitory sequence encodesan inhibitory nucleotide molecule (for example, hairpin RNA, miRNA, ordouble-stranded RNA polynucleotides), or encodes an inhibitorypolypeptide (e.g., a polypeptide that inhibits expression or function ofthe target gene product), following its transcription (for example, inthe case of an inhibitory sequence encoding a hairpin RNA, miRNA, ordouble-stranded RNA polynucleotide) or its transcription and translation(in the case of an inhibitory sequence encoding an inhibitorypolypeptide), the transcribed or translated product, respectively,exerts the inhibitory effect on the target gene product (e.g., inhibitsexpression or function of the target gene product).

A ODC inhibitory sequence disclosed can be a sequence triggering genesilencing via any silencing pathway or mechanism known in the art,including, but not limited to, sense suppression/cosuppression,antisense suppression, double-stranded RNA (dsRNA) interference, hairpinRNA interference and intron-containing hairpin RNA interference,amplicon-mediated interference, ribozymes, small interfering RNA,artificial or synthetic microRNA, and artificial trans-acting siRNA. AnODC inhibitory sequence may range from at least about 20 nucleotides,about 50 nucleotides, about 70 nucleotides, about 100 nucleotides, about150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 350 nucleotides, about 400 nucleotides, and up to thefull-length polynucleotide encoding the proteins of the presentdisclosure, depending upon the desired outcome. In an aspect, a ODCinhibitory sequence can be a fragment of between about 50 and about 400nucleotides, between about 70 and about 350 nucleotides, between about90 and about 325 nucleotides, between about 90 and about 300nucleotides, between about 90 and about 275 nucleotides, between about100 and about 400 nucleotides, between about 100 and about 350nucleotides, between about 100 and about 325 nucleotides, between about100 and about 300 nucleotides, between about 125 and about 300nucleotides, or between about 125 and about 275 nucleotides in length.

The use of the term “polynucleotide” is not intended to limit thepresent disclosure to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the present disclosure also encompass all forms ofsequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

In an aspect, the present disclosure provides recombinant DNA constructscomprising a promoter that is functional in a tobacco cell and operablylinked to a polynucleotide that encodes an RNA molecule capable ofbinding to an RNA encoding a polypeptide having an amino acid sequenceat least 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 23 to 28, and fragmentsthereof, and where the RNA molecule suppresses the expression of thepolypeptide. In an aspect, the RNA molecule is selected from the groupconsisting of a microRNA, an siRNA, and a trans-acting siRNA. In anotheraspect, the recombinant DNA construct encodes a double stranded RNA.Also provided are transgenic tobacco plants or part thereof, curedtobacco material, or tobacco products comprising these recombinant DNAconstructs. In an aspect, these transgenic plants, cured tobaccomaterial, or tobacco products comprise a lower level of nicotinecompared to a control tobacco plant without the recombinant DNAconstruct. Further provided are methods of reducing the nicotine levelof a tobacco plant, the method comprising transforming a tobacco plantwith any of these recombinant DNA constructs.

As used herein, “operably linked” refers to a functional linkage betweentwo or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (e.g., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.

As used herein and when used in reference to a sequence, “heterologous”refers to a sequence that originates from a foreign species, or, if fromthe same species, is substantially modified from its native form incomposition and/or genomic location by deliberate human intervention.The term also is applicable to nucleic acid constructs, also referred toherein as “polynucleotide constructs” or “nucleotide constructs.” Inthis manner, a “heterologous” nucleic acid construct is intended to meana construct that originates from a foreign species, or, if from the samespecies, is substantially modified from its native form in compositionand/or genomic location by deliberate human intervention. Heterologousnucleic acid constructs include, but are not limited to, recombinantnucleotide constructs that have been introduced into a plant or plantpart thereof, for example, via transformation methods or subsequentbreeding of a transgenic plant with another plant of interest. In anaspect, a promoter used is heterologous to the sequence driven by thepromoter. In another aspect, a promoter used is heterologous to tobacco.In a further aspect, a promoter used is native to tobacco.

In an aspect, a modified tobacco plant described is a cisgenic plant. Asused herein, “cisgenesis” or “cisgenic” refers to genetic modificationof a plant, plant cell, or plant genome in which all components (e.g.,promoter, donor nucleic acid, selection gene) have only plant origins(i.e., no non-plant origin components are used). In an aspect, amodified plant, plant cell, or plant genome provided is cisgenic.Cisgenic plants, plant cells, and plant genomes provided can lead toready-to-use tobacco lines. In another aspect, a modified tobacco plantprovided comprises no non-tobacco genetic material or sequences.

As used herein, “gene expression” refers to the biosynthesis orproduction of a gene product, including the transcription and/ortranslation of the gene product.

In an aspect, recombinant DNA constructs or expression cassettes canalso comprise a selectable marker gene for the selection of transgeniccells. Selectable marker genes include, but are not limited to, genesencoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP).

In an aspect, recombinant DNA constructs or expression cassettescomprise a promoter selected from the group consisting of a constitutivepromoter, an inducible promoter, and a tissue-preferred promoter (forexample, a leaf-specific or root-specific promoter). Exemplaryconstitutive promoters include the core promoter of the Rsyn7 promoterand other constitutive promoters disclosed in U.S. Pat. No. 6,072,050;the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Exemplary chemical-inducible promoters include the tobacco PR-lapromoter, which is activated by salicylic acid. Other chemical-induciblepromoters of interest include steroid-responsive promoters (see, forexample, the glucocorticoid-inducible promoter in Schena et al. (1991)Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998)Plant J. 14(2):247-257) and tetracycline-inducible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156). Additional exemplary promoters that canbe used are those responsible for heat-regulated gene expression,light-regulated gene expression (for example, the pea rbcS-3A; the maizerbcS promoter; the chlorophyll alb-binding protein gene found in pea; orthe Arabssu promoter), hormone-regulated gene expression (for example,the abscisic acid (ABA) responsive sequences from the Em gene of wheat;the ABA-inducible HVA1 and HVA22, and rd29A promoters of barley andArabidopsis; and wound-induced gene expression (for example, of wunl),organ specific gene expression (for example, of the tuber-specificstorage protein gene; the 23-kDa zein gene from maize described by; orthe French bean (β-phaseolin gene), or pathogen-inducible promoters (forexample, the PR-1, prp-1, or (β-1,3 glucanase promoters, thefungal-inducible wir1a promoter of wheat, and the nematode-induciblepromoters, TobRB7-5A and Hmg-1, of tobacco arid parsley, respectively).

In an aspect, a tobacco plant provided further comprises increased orreduced expression of activity of genes involved in nicotinebiosynthesis or transport. Genes involved in nicotine biosynthesisinclude, but are not limited to, arginine decarboxylase (ADC),methylputrescine oxidase (MPO), NADH dehydrogenase, ornithinedecarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthetase (SAMS). NicotineSynthase, which catalyzes the condensation step between a nicotinic acidderivative and methylpyrrolinium cation, has not been elucidatedalthough two candidate genes (A622 and NBB1) have been proposed. See US2007/0240728 A1 and US 2008/0120737A1. A622 encodes an isoflavonereductase-like protein. In addition, several transporters may beinvolved in the translocation of nicotine. A transporter gene, namedMATE, has been cloned and characterized (Morita et al., PNAS 106:2447-52(2009)).

In an aspect, a tobacco plant provided further comprises an increased orreduced level of mRNA, protein, or both of one or more genes encoding aproduct selected from the group consisting of PMT, MPO, QPT, ADC, ODC,PRAI, SAMS, BBL, MATE, A622, and NBB1, compared to a control tobaccoplant. In another aspect, a tobacco plants provided further comprises atransgene directly suppressing the expression of one or more genesencoding a product selected from the group consisting of PMT, MPO, QPT,ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, atobacco plant provided further comprises a transgene or mutationsuppressing the expression or activity of one or more genes encoding aproduct selected from the group consisting of PMT, MPO, QPT, ADC, ODC,PRAI, SAMS, BBL, MATE, A622, and NBB1. In another aspect, a tobaccoplant provided further comprises a transgene overexpressing one or moregenes encoding a product selected from the group consisting of PMT, MPO,QPT, ADC, ODC, PRAI, SAMS, BBL, MATE, A622, and NBB1.

Also disclosed are the transformation of tobacco plants with recombinantconstructs or expression cassettes described using any suitabletransformation methods known in the art. Methods for introducingpolynucleotide sequences into tobacco plants are known in the art andinclude, but are not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods. “Stabletransformation” refers to transformation where the nucleotide constructof interest introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a sequence isintroduced into the plant and is only temporally expressed or is onlytransiently present in the plant.

Suitable methods of introducing polynucleotides into plant cells of thepresent disclosure include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Shillito et al. (1987) Meth.Enzymol. 153:313-336; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos.5,104,310, 5,149,645, 5,177,010, 5,231,019, 5,463,174, 5,464,763,5,469,976, 4,762,785, 5,004,863, 5,159,135, 5,563,055, and 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. Nos.4,945,050, 5,141,131, 5,886,244, 5,879,918, and 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed.Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev.Genet. 22:421-477; Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues,ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler etal. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992)Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation);D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation).

In another aspect, recombinant constructs or expression cassettes may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anexpression cassette of the present disclosure within a viral DNA or RNAmolecule. It is recognized that promoters for use in expressioncassettes also encompass promoters utilized for transcription by viralRNA polymerases. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221.

Any plant tissue that can be subsequently propagated using clonalmethods, whether by organogenesis or embryogenesis, may be transformedwith a recombinant construct or an expression cassette. By“organogenesis” in intended the process by which shoots and roots aredeveloped sequentially from meristematic centers. By “embryogenesis” isintended the process by which shoots and roots develop together in aconcerted fashion (not sequentially), whether from somatic cells orgametes. Exemplary tissues that are suitable for various transformationprotocols described include, but are not limited to, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems) and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem), hypocotyls, cotyledons, leaf disks, pollen,embryos, and the like.

In an aspect, a tobacco plant provided is from a tobacco type selectedfrom the group consisting of flue-cured tobacco, air-cured tobacco, darkair-cured tobacco, dark fire-cured tobacco, Galpao tobacco, and Orientaltobacco. In another aspect, a tobacco plant provided is from a tobaccotype selected from the group consisting of Burley tobacco, Marylandtobacco, and dark tobacco.

In an aspect, a tobacco plant provided is in a flue-cured tobaccobackground or exhibits one or more flue-cured tobacco characteristicdescribed here. Flue-cured tobaccos (also called Virginia or brighttobaccos) amount to approximately 40% of world tobacco production.Flue-cured tobaccos are often also referred to as “bright tobacco”because of the golden-yellow to deep-orange color it reaches duringcuring. Flue-cured tobaccos have a light, bright aroma and taste.Flue-cured tobaccos are generally high in sugar and low in oils. Majorflue-cured tobacco growing countries are Argentina, Brazil, China,India, Tanzania and the U.S. In an aspect, a low-alkaloid orlow-nicotine tobacco plant or seed provided is in a flue-cured tobaccobackground selected from the group consisting of CC 13, CC 27, CC 33, CC37, CC 65, CC 67, CC 700, GF 318, GL 338, GL 368, GL 939, K 346, K 399,K326, NC 102, NC 196, NC 291, NC 297, NC 299, NC 471, NC 55, NC 606, NC71, NC 72, NC 92, PVH 1118, PVH 1452, PVH 2110, SPEIGHT 168, SPEIGHT220, SPEIGHT 225, SPEIGHT 227, SPEIGHT 236, and any variety essentiallyderived from any one of the foregoing varieties. In another aspect, alow-alkaloid or low-nicotine tobacco plant or seed provided is in aflue-cured tobacco background selected from the group consisting ofCoker 48, Coker 176, Coker 371-Gold, Coker 319, Coker 347, GL 939, K149, K326, K 340, K 346, K 358, K 394, K 399, K 730, NC 27NF, NC 37NF,NC 55, NC 60, NC 71, NC 72, NC 82, NC 95, NC 297, NC 606, NC 729, NC2326, McNair 373, McNair 944, Ox 207, Ox 414 NF, Reams 126, Reams 713,Reams 744, RG 8, RG 11, RG 13, RG 17, RG 22, RG 81, RG H4, RG H51,Speight H-20, Speight G-28, Speight G-58, Speight G-70, Speight G-108,Speight G-111, Speight G-117, Speight 168, Speight 179, Speight NF-3, Va116, Va 182, and any variety essentially derived from any one of theforegoing varieties. See WO 2004/041006 A1. In a further aspect,low-alkaloid or low-nicotine tobacco plants, seeds, hybrids, varieties,or lines are in any flue cured background selected from the groupconsisting of K326, K346, and NC196.

In an aspect, a tobacco plant provided is in an air-cured tobaccobackground or exhibits one or more air-cured tobacco characteristicdescribed here. Air-cured tobaccos include Burley, Md., and darktobaccos. The common factor is that curing is primarily withoutartificial sources of heat and humidity. Burley tobaccos are light todark brown in color, high in oil, and low in sugar. Burley tobaccos areair-cured in barns. Major Burley growing countries are Argentina,Brazil, Italy, Malawi, and the U.S. Maryland tobaccos are extremelyfluffy, have good burning properties, low nicotine and a neutral aroma.Major Maryland growing countries include the U.S. and Italy. In anaspect, a low-alkaloid or low-nicotine tobacco plant or seed provided isin a Burley tobacco background selected from the group consisting ofClay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R 711, R 712,NCBH 129, Bu 21×Ky 10, HBO4P, Ky 14×L 8, Kt 200, Newton 98, Pedigo 561,Pf561 and Va 509. In a further aspect, low-alkaloid or low-nicotinetobacco plants, seeds, hybrids, varieties, or lines are in any Burleybackground selected from the group consisting of TN 90, KT 209, KT 206,KT212, and HB 4488. In another aspect, a low-alkaloid or low-nicotinetobacco plant or seed provided is in a Maryland tobacco backgroundselected from the group consisting of Md 10, Md 40, Md 201, Md 609, Md872 and Md 341.

In an aspect, a tobacco plant provided is in a dark air-cured tobaccobackground or exhibits one or more dark air-cured tobacco characteristicdescribed here. Dark air-cured tobaccos are distinguished from othertypes primarily by its curing process which gives dark air-cured tobaccoits medium- to dark-brown color and distinct aroma. Dark air-curedtobaccos are mainly used in the production of chewing tobacco and snuff.In an aspect, a low-alkaloid or low-nicotine tobacco plant or seedprovided is in a dark air-cured tobacco background selected from thegroup consisting of Sumatra, Jatim, Dominican Cubano, Besuki, Onesucker, Green River, Va. sun-cured, and Paraguan Passado.

In an aspect, a tobacco plant provided is in a dark fire-cured tobaccobackground or exhibits one or more dark fire-cured tobaccocharacteristic described here. Dark fire-cured tobaccos are generallycured with low-burning wood fires on the floors of closed curing barns.Their leaves have low sugar content but high nicotine content. Darkfire-cured tobaccos are used for making pipe blends, cigarettes, chewingtobacco, snuff and strong-tasting cigars. Major growing regions for darkfire-cured tobaccos are Tennessee, Kentucky, and Virginia, USA. In anaspect, a low-alkaloid or low-nicotine tobacco plant or seed provided isin a dark fire-cured tobacco background selected from the groupconsisting of Narrow Leaf Madole, Improved Madole, Tom Rosson Madole,Newton's VH Madole, Little Crittenden, Green Wood, Little Wood, SmallStalk Black Mammoth, DT 508, DT 518, DT 592, KY 171, DF 911, DF 485, TND94, TN D950, VA 309, and VA 359.

In an aspect, a tobacco plant provided is in an Oriental tobaccobackground or exhibits one or more Oriental tobacco characteristicdescribed here. Oriental tobaccos are also referred to as Greek, aromaand Turkish tobaccos due to the fact that they are typically grown ineastern Mediterranean regions such as Turkey, Greece, Bulgaria,Macedonia, Syria, Lebanon, Italy, and Romania. The small plant and leafsize, characteristic of today's Oriental varieties, as well as itsunique aroma properties are a result of the plant's adaptation to thepoor soil and stressful climatic conditions in which it develop overmany past centuries. In an aspect, a low-alkaloid or low-nicotinetobacco plant or seed provided is in an Oriental tobacco backgroundselected from the group consisting of Izmir, Katerini, Samsun, Basma andKrumovgrad, Trabzon, Thesalian, Tasova, Sinop, Izmit, Hendek, Edirne,Semdinli, Adiyanman, Yayladag, Iskenderun, Duzce, Macedonian, Mavra,Prilep, Bafra, Bursa, Bucak, Bitlis, Balikesir, and any varietyessentially derived from any one of the foregoing varieties.

In an aspect, low-alkaloid or low-nicotine tobacco plants, seeds,hybrids, varieties, or lines are essentially derived from or in thegenetic background of BU 64, CC 101, CC 200, CC 27, CC 301, CC 400, CC500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 350, GL 600,GL 737, GL 939, GL 973, HB 04P, K 149, K 326, K 346, K 358, K394, K 399,K 730, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171, KY907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944,msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297,NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (TomRosson) Madole, VA 309, or VA359, Maryland 609, HB3307PLC, HB4488PLC,KT206LC, KT209LC, KT210LC, KT212LC, R610LC, PVH2310, NC196, KTD14LC,KTD6LC, KTD8LC, PD7302LC, PD7305LC, PD7309LC, PD7318LC, PD7319LC,PD7312LC, ShireyLC, or any commercial tobacco variety according tostandard tobacco breeding techniques known in the art.

All foregoing mentioned specific varieties of dark air-cured, Burley,Md., dark fire-cured, or Oriental type are listed only for exemplarypurposes. Any additional dark air-cured, Burley, Md., dark fire-cured,Oriental varieties are also contemplated in the present application.

Also provided are populations of tobacco plants described. In an aspect,a population of tobacco plants has a planting density of between about5,000 and about 8000, between about 5,000 and about 7,600, between about5,000 and about 7,200, between about 5,000 and about 6,800, betweenabout 5,000 and about 6,400, between about 5,000 and about 6,000,between about 5,000 and about 5,600, between about 5,000 and about5,200, between about 5,200 and about 8,000, between about 5,600 andabout 8,000, between about 6,000 and about 8,000, between about 6,400and about 8,000, between about 6,800 and about 8,000, between about7,200 and about 8,000, or between about 7,600 and about 8,000 plants peracre. In another aspect, a population of tobacco plants is in a soiltype with low to medium fertility.

Also provided are containers of seeds from tobacco plants described. Acontainer of tobacco seeds of the present disclosure may contain anynumber, weight, or volume of seeds. For example, a container can containat least, or greater than, about 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or more seeds.Alternatively, the container can contain at least, or greater than,about 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4pounds, 5 pounds or more seeds. Containers of tobacco seeds may be anycontainer available in the art. By way of non-limiting example, acontainer may be a box, a bag, a packet, a pouch, a tape roll, a tube,or a bottle.

Also provided is cured tobacco material made from a low-alkaloid orlow-nicotine tobacco plant described. Further provided is cured tobaccomaterial made from a tobacco plant described with higher levels of totalalkaloid or nicotine.

“Curing” is the aging process that reduces moisture and brings about thedestruction of chlorophyll giving tobacco leaves a golden color and bywhich starch is converted to sugar. Cured tobacco therefore has a higherreducing sugar content and a lower starch content compared to harvestedgreen leaf. In an aspect, green leaf tobacco provided can be cured usingconventional means, e.g., flue-cured, barn-cured, fire-cured, air-curedor sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford) for a description of different types of curingmethods. Cured tobacco is usually aged in a wooden drum (e.g., ahogshead) or cardboard cartons in compressed conditions for severalyears (e.g., two to five years), at a moisture content ranging from 10%to about 25%. See, U.S. Pat. Nos. 4,516,590 and 5,372,149. Cured andaged tobacco then can be further processed. Further processing includesconditioning the tobacco under vacuum with or without the introductionof steam at various temperatures, pasteurization, and fermentation.Fermentation typically is characterized by high initial moisturecontent, heat generation, and a 10 to 20% loss of dry weight. See, e.g.,U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S.Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cure, aged, and fermented tobacco can be furtherprocessed (e.g., cut, shredded, expanded, or blended). See, for example,U.S. Pat. Nos. 4,528,993; 4,660,577; and 4,987,907. In an aspect, thecured tobacco material of the present disclosure is sun-cured. Inanother aspect, the cured tobacco material of the present disclosure isflue-cured, air-cured, or fire-cured.

Tobacco material obtained from the tobacco lines, varieties or hybridsof the present disclosure can be used to make tobacco products. As usedherein, “tobacco product” is defined as any product made or derived fromtobacco that is intended for human use or consumption.

Tobacco products provided include, without limitation, cigaretteproducts (e.g., cigarettes and bidi cigarettes), cigar products (e.g.,cigar wrapping tobacco and cigarillos), pipe tobacco products, productsderived from tobacco, tobacco-derived nicotine products, smokelesstobacco products (e.g., moist snuff, dry snuff, and chewing tobacco),films, chewables, tabs, shaped parts, gels, consumable units, insolublematrices, hollow shapes, reconstituted tobacco, expanded tobacco, andthe like. See, e.g., U.S. Patent Publication No. US 2006/0191548.

As used herein, “cigarette” refers a tobacco product having a “rod” and“filler”. The cigarette “rod” includes the cigarette paper, filter, plugwrap (used to contain filtration materials), tipping paper that holdsthe cigarette paper (including the filler) to the filter, and all gluesthat hold these components together. The “filler” includes (1) alltobaccos, including but not limited to reconstituted and expandedtobacco, (2) non-tobacco substitutes (including but not limited toherbs, non-tobacco plant materials and other spices that may accompanytobaccos rolled within the cigarette paper), (3) casings, (4)flavorings, and (5) all other additives (that are mixed into tobaccosand substitutes and rolled into the cigarette).

As used herein, “reconstituted tobacco” refers to a part of tobaccofiller made from tobacco dust and other tobacco scrap material,processed into sheet form and cut into strips to resemble tobacco. Inaddition to the cost savings, reconstituted tobacco is very importantfor its contribution to cigarette taste from processing flavordevelopment using reactions between ammonia and sugars.

As used herein, “expanded tobacco” refers to a part of tobacco fillerwhich is processed through expansion of suitable gases so that thetobacco is “puffed” resulting in reduced density and greater fillingcapacity. It reduces the weight of tobacco used in cigarettes.

Tobacco products derived from plants of the present disclosure alsoinclude cigarettes and other smoking articles, particularly thosesmoking articles including filter elements, where the rod of smokablematerial includes cured tobacco within a tobacco blend. In an aspect, atobacco product of the present disclosure is selected from the groupconsisting of a cigarillo, a non-ventilated recess filter cigarette, avented recess filter cigarette, a cigar, snuff, pipe tobacco, cigartobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookahtobacco, shredded tobacco, and cut tobacco. In another aspect, a tobaccoproduct of the present disclosure is a smokeless tobacco product.Smokeless tobacco products are not combusted and include, but notlimited to, chewing tobacco, moist smokeless tobacco, snus, and drysnuff. Chewing tobacco is coarsely divided tobacco leaf that istypically packaged in a large pouch-like package and used in a plug ortwist. Moist smokeless tobacco is a moist, more finely divided tobaccothat is provided in loose form or in pouch form and is typicallypackaged in round cans and used as a pinch or in a pouch placed betweenan adult tobacco consumer's cheek and gum. Snus is a heat treatedsmokeless tobacco. Dry snuff is finely ground tobacco that is placed inthe mouth or used nasally. In a further aspect, a tobacco product of thepresent disclosure is selected from the group consisting of loose leafchewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff. Inyet another aspect, a tobacco product of the present disclosure isselected from the group consisting of an electronically heatedcigarette, an e-cigarette, an electronic vaporing device.

In an aspect, a tobacco product of the present disclosure can be ablended tobacco product. In one aspect, a blended tobacco productcomprises cured tobacco materials. In an aspect, a cured tobaccomaterial constitutes about at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofcured tobacco in a tobacco blend by weight. In one aspect, a curedtobacco material constitutes about at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% of cured tobacco in a tobacco blend by volume.

In an aspect, a tobacco product of the present disclosure can be a lownicotine tobacco product. In a further aspect, a tobacco product of thepresent disclosure may comprise nornicotine at a level of less thanabout 3 mg/g. For example, the nornicotine content in such a product canbe about 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 pg/g, 500pg/g, 250 pg/g, 100 pg/g, 75 pg/g, 50 pg/g, 25 pg/g, 10 pg/g, 7.0 pg/g,5.0 pg/g, 4.0 pg/g, 2.0 pg/g, 1.0 pg/g, 0.5 pg/g, 0.4 pg/g, 0.2 pg/g,0.1 pg/g, 0.05 pg/g, 0.01 pg/g, or undetectable.

In an aspect, cured tobacco material or tobacco products providedcomprise an average nicotine or total alkaloid level selected from thegroup consisting of about 0.01%, 0.02%, 0.05%, 0.75%, 0.1%, 0.15%, 0.2%,0.3%, 0.35%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, and 9% on a dry weight basis. In anotheraspect, cured tobacco material or tobacco products provided comprise anaverage nicotine or total alkaloid level selected from the groupconsisting of about between 0.01% and 0.02%, between 0.02% and 0.05%,between 0.05% and 0.75%, between 0.75% and 0.1%, between 0.1% and 0.15%,between 0.15% and 0.2%, between 0.2% and 0.3%, between 0.3% and 0.35%,between 0.35% and 0.4%, between 0.4% and 0.5%, between 0.5% and 0.6%,between 0.6% and 0.7%, between 0.7% and 0.8%, between 0.8% and 0.9%,between 0.9% and 1%, between 1% and 1.1%, between 1.1% and 1.2%, between1.2% and 1.3%, between 1.3% and 1.4%, between 1.4% and 1.5%, between1.5% and 1.6%, between 1.6% and 1.7%, between 1.7% and 1.8%, between1.8% and 1.9%, between 1.9% and 2%, between 2% and 2.1%, between 2.1%and 2.2%, between 2.2% and 2.3%, between 2.3% and 2.4%, between 2.4% and2.5%, between 2.5% and 2.6%, between 2.6% and 2.7%, between 2.7% and2.8%, between 2.8% and 2.9%, between 2.9% and 3%, between 3% and 3.1%,between 3.1% and 3.2%, between 3.2% and 3.3%, between 3.3% and 3.4%,between 3.4% and 3.5%, and between 3.5% and 3.6% on a dry weight basis.In a further aspect, cured tobacco material or tobacco products providedcomprise an average nicotine or total alkaloid level selected from thegroup consisting of about between 0.01% and 0.1%, between 0.02% and0.2%, between 0.03% and 0.3%, between 0.04% and 0.4%, between 0.05% and0.5%, between 0.75% and 1%, between 0.1% and 1.5%, between 0.15% and 2%,between 0.2% and 3%, and between 0.3% and 3.5% on a dry weight basis.

The present disclosure also provides methods for breeding tobacco lines,cultivars, or varieties comprising a desirable level of total alkaloidor nicotine, e.g., low nicotine or nicotine free. Breeding can becarried out via any known procedures. DNA fingerprinting, SNP mapping,haplotype mapping or similar technologies may be used in amarker-assisted selection (MAS) breeding program to transfer or breed adesirable trait or allele into a tobacco plant. For example, a breedercan create segregating populations in a F₂ or backcross generation usingF1 hybrid plants or further crossing the F1 hybrid plants with otherdonor plants with an agronomically desirable genotype. Plants in the F₂or backcross generations can be screened for a desired agronomic traitor a desirable chemical profile using one of the techniques known in theart or listed herein. Depending on the expected inheritance pattern orthe MAS technology used, self-pollination of selected plants before eachcycle of backcrossing to aid identification of the desired individualplants can be performed. Backcrossing or other breeding procedure can berepeated until the desired phenotype of the recurrent parent isrecovered. A recurrent parent in the present disclosure can be aflue-cured variety, a Burley variety, a dark air-cured variety, a darkfire-cured variety, or an Oriental variety. Other breeding techniquescan be found, for example, in Wernsman, E. A., and Rufty, R. C. 1987.Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. CropSpecies. W. H. Fehr (ed.), MacMillan Publishing Go, Inc., New York,N.Y., incorporated herein by reference in their entirety.

Results of a plant breeding program using the tobacco plants describedincludes useful lines, cultivars, varieties, progeny, inbreds, andhybrids of the present disclosure. As used herein, the term “variety”refers to a population of plants that share constant characteristicswhich separate them from other plants of the same species. A variety isoften, although not always, sold commercially. While possessing one ormore distinctive traits, a variety is further characterized by a verysmall overall variation between individuals within that variety. A “pureline” variety may be created by several generations of self-pollinationand selection, or vegetative propagation from a single parent usingtissue or cell culture techniques. A variety can be essentially derivedfrom another line or variety. As defined by the International Conventionfor the Protection of New Varieties of Plants (Dec. 2, 1961, as revisedat Geneva on Nov. 10, 1972; on Oct. 23, 1978; and on Mar. 19, 1991), avariety is “essentially derived” from an initial variety if: a) it ispredominantly derived from the initial variety, or from a variety thatis predominantly derived from the initial variety, while retaining theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety; b) it isclearly distinguishable from the initial variety; and c) except for thedifferences which result from the act of derivation, it conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety. Essentially derived varieties can be obtained, for example, bythe selection of a natural or induced mutant, a somaclonal variant, avariant individual from plants of the initial variety, backcrossing, ortransformation. A first tobacco variety and a second tobacco varietyfrom which the first variety is essentially derived, are considered ashaving essentially identical genetic background. A “line” asdistinguished from a variety most often denotes a group of plants usednon-commercially, for example in plant research. A line typicallydisplays little overall variation between individuals for one or moretraits of interest, although there may be some variation betweenindividuals for other traits.

As used herein, “locus” is a chromosome region where a polymorphicnucleic acid, trait determinant, gene, or marker is located. The loci ofthis disclosure comprise one or more polymorphisms in a population;e.g., alternative alleles are present in some individuals. As usedherein, “allele” refers to an alternative nucleic acid sequence at aparticular locus. The length of an allele can be as small as 1nucleotide base, but is typically larger. For example, a first allelecan occur on one chromosome, while a second allele occurs on a secondhomologous chromosome, e.g., as occurs for different chromosomes of aheterozygous individual, or between different homozygous or heterozygousindividuals in a population. As used herein, the term “chromosomeinterval” designates a contiguous linear span of genomic DNA thatresides on a single chromosome.

As used herein, “introgression” or “introgress” refers to thetransmission of a desired allele of a genetic locus from one geneticbackground to another.

As used herein, “crossed” or “cross” means to produce progeny viafertilization (e.g. cells, seeds or plants) and includes crosses betweenplants (sexual) and self fertilization (selfing).

As used herein, “backcross” and “backcrossing” refer to the processwhereby a progeny plant is repeatedly crossed back to one of itsparents. In a backcrossing scheme, the “donor” parent refers to theparental plant with the desired gene or locus to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. The initial cross gives rise to the F1generation. The term “BC1” refers to the second use of the recurrentparent, “BC2” refers to the third use of the recurrent parent, and soon. In an aspect, a backcross is performed repeatedly, with a progenyindividual of each successive backcross generation being itselfbackcrossed to the same parental genotype.

As used herein, “single gene converted” or “single gene conversion”refers to plants that are developed using a plant breeding techniqueknown as backcrossing, or via genetic engineering, where essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety via the backcrossing technique or via genetic engineering.

As used herein, “elite variety” means any variety that has resulted frombreeding and selection for superior agronomic performance.

As used herein, “selecting” or “selection” in the context ofmarker-assisted selection or breeding refer to the act of picking orchoosing desired individuals, normally from a population, based oncertain pre-determined criteria.

As used herein, the term “trait” refers to one or more detectablecharacteristics of a cell or organism which can be influenced bygenotype. The phenotype can be observable to the naked eye, or by anyother means of evaluation known in the art, e.g., microscopy,biochemical analysis, genomic analysis, an assay for a particulardisease tolerance, etc. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, e.g., a “single genetrait.” In other cases, a phenotype is the result of several genes.

It is understood that any tobacco plant of the present disclosure canfurther comprise additional agronomically desirable traits, for example,by transformation with a genetic construct or transgene using atechnique known in the art. Without limitation, an example of a desiredtrait is herbicide resistance, pest resistance, disease resistance; highyield; high grade index value; curability; curing quality; mechanicalharvestability; holding ability; leaf quality; height, plant maturation(e.g., early maturing, early to medium maturing, medium maturing, mediumto late maturing, or late maturing); stalk size (e.g., a small, medium,or a large stalk); or leaf number per plant (e.g., a small (e.g., 5-10leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number ofleaves), or any combination. In an aspect, low-nicotine or nicotine-freetobacco plants or seeds disclosed comprise one or more transgenesexpressing one or more insecticidal proteins, such as, for example, acrystal protein of Bacillus thuringiensis or a vegetative insecticidalprotein from Bacillus cereus, such as VIP3 (see, for example, Estruch etal. (1997) Nat. Biotechnol.15:137). In another aspect, tobacco plantsfurther comprise an introgressed trait conferring resistance to brownstem rot (U.S. Pat. No. 5,689,035) or resistance to cyst nematodes (U.S.Pat. No. 5,491,081).

The present disclosure also provides tobacco plants comprising analtered nicotine or total alkaloid level but having a yield comparableto the yield of corresponding initial tobacco plants without such anicotine level alternation. In an aspect, a low-nicotine ornicotine-free tobacco variety provides a yield selected from the groupconsisting of about between 1200 and 3500, between 1300 and 3400,between 1400 and 3300, between 1500 and 3200, between 1600 and 3100,between 1700 and 3000, between 1800 and 2900, between 1900 and 2800,between 2000 and 2700, between 2100 and 2600, between 2200 and 2500, andbetween 2300 and 2400 lbs/acre. In another aspect, a low-nicotine ornicotine-free tobacco variety provides a yield selected from the groupconsisting of about between 1200 and 3500, between 1300 and 3500,between 1400 and 3500, between 1500 and 3500, between 1600 and 3500,between 1700 and 3500, between 1800 and 3500, between 1900 and 3500,between 2000 and 3500, between 2100 and 3500, between 2200 and 3500,between 2300 and 3500, between 2400 and 3500, between 2500 and 3500,between 2600 and 3500, between 2700 and 3500, between 2800 and 3500,between 2900 and 3500, between 3000 and 3500, and between 3100 and 3500lbs/acre. In a further aspect, low-nicotine or nicotine-free tobaccoplants provide a yield between 65% and 130%, between 70% and 130%,between 75% and 130%, between 80% and 130%, between 85% and 130%,between 90% and 130%, between 95% and 130%, between 100% and 130%,between 105% and 130%, between 110% and 130%, between 115% and 130%, orbetween 120% and 130% of the yield of a control plant having essentiallyidentical genetic background except a nic1b mutation, a nic2 mutation, aNic1b transgene, a Nic2 transgene, or combinations thereof. In a furtheraspect, low-nicotine or nicotine-free tobacco plants provide a yieldbetween 70% and 125%, between 75% and 120%, between 80% and 115%,between 85% and 110%, or between 90% and 100% of the yield of a controlplant having essentially identical genetic background except a nic1mutation, a nic2 mutation, a Nic1 transgene, a Nic2 transgene, orcombinations thereof.

In an aspect, a tobacco plant (e.g., a low-nicotine, nicotine-free, orlow-alkaloid tobacco variety) does not exhibit one or more, two or more,three or more, or all of the LA BU21 traits selected from the groupconsisting of lower yield, delayed ripening and senescence, highersusceptibility to insect herbivory, increased polyamine content aftertopping, higher chlorophyll, more mesophyll cells per unit leaf area,and poor end-product quality after curing. In an aspect, a tobacco plantdisclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobaccovariety) does not exhibit two or more of the LA BU21 traits selectedfrom the group consisting of lower yield, delayed ripening andsenescence, higher susceptibility to insect herbivory, increasedpolyamine content after topping, higher chlorophyll, more mesophyllcells per unit leaf area, and poor end-product quality after curing. Inan aspect, a tobacco plant disclosed (e.g., a low-nicotine,nicotine-free, or low-alkaloid tobacco variety) does not exhibit threeor more of the LA BU21 traits selected from the group consisting oflower yield, delayed ripening and senescence, higher susceptibility toinsect herbivory, increased polyamine content after topping, higherchlorophyll, more mesophyll cells per unit leaf area, and poorend-product quality after curing. In an aspect, a tobacco plantdisclosed (e.g., a low-nicotine, nicotine-free, or low-alkaloid tobaccovariety) exhibits at a lower level compared to LA BU21, LAFC53, or LNKY171, one or more, two or more, three or more, or all of the LA BU21traits selected from the group consisting of lower yield, delayedripening and senescence, higher susceptibility to insect herbivory,increased polyamine content after topping, higher chlorophyll, moremesophyll cells per unit leaf area, and poor end-product quality aftercuring. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine,nicotine-free, or low-alkaloid tobacco variety) exhibits at a lowerlevel compared to LA BU21, LAFC53, or LN KY171, two or more of the LABU21 traits selected from the group consisting of lower yield, delayedripening and senescence, higher susceptibility to insect herbivory,increased polyamine content after topping, higher chlorophyll, moremesophyll cells per unit leaf area, and poor end-product quality aftercuring. In an aspect, a tobacco plant disclosed (e.g., a low-nicotine,nicotine-free, or low-alkaloid tobacco variety) exhibits at a lowerlevel compared to LA BU21, LAFC53, or LN KY171, three or more, or all ofthe LA BU21 traits selected from the group consisting of lower yield,delayed ripening and senescence, higher susceptibility to insectherbivory, increased polyamine content after topping, higherchlorophyll, more mesophyll cells per unit leaf area, and poorend-product quality after curing.

In an aspect, a modified tobacco plant (e.g., a low-nicotine,nicotine-free, or low-alkaloid tobacco variety) comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) without substantially impacting a trait selected from thegroup consisting of yield, ripening and senescence, susceptibility toinsect herbivory, polyamine content after topping, chlorophyll level,mesophyll cell number per unit leaf area, and end-product quality aftercuring.

In an aspect, a modified tobacco plant comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a trait substantially comparable toan unmodified control plant, where the trait is selected from the groupconsisting of yield, ripening and senescence, susceptibility to insectherbivory, polyamine content after topping, chlorophyll level, mesophyllcell number per unit leaf area, and end-product quality after curing.

In an aspect, a modified tobacco plant comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a yield which is more than 80%, morethan 85%, more than 90%, more than 95%, more than 100%, more than 105%,more than 110%, more than 115%, more than 120%, more than 125%, morethan 130%, more than 135%, or more than 140% relative to the yield of anunmodified control plant. In an aspect, a modified tobacco plantdisclosed comprises a modification conferring a desired trait (e.g.,low-nicotine, nicotine-free, or low-alkaloid) and further comprises ayield which is between 70% and 140%, between 75% and 135%, between 80%and 130%, between 85% and 125%, between 90% and 120%, between 95% and115%, or between 100% and 110% relative to the yield of an unmodifiedcontrol plant. In an aspect, a modified tobacco plant disclosedcomprises a modification conferring a desired trait (e.g., low-nicotine,nicotine-free, or low-alkaloid) and further comprises a yield which isbetween 70% and 80%, between 75% and 85%, between 80% and 90%, between85% and 95%, between 90% and 100%, between 95% and 105%, between 105%and 115%, between 110% and 120%, between 115% to 125%, between 120% and130%, between 125 and 135%, or between 130% and 140% relative to theyield of an unmodified control plant.

In an aspect, a modified tobacco plant comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a polyamine content after toppingwhich is more than 80%, more than 85%, more than 90%, more than 95%,more than 100%, more than 105%, more than 110%, more than 115%, morethan 120%, more than 125%, more than 130%, more than 135%, or more than140% relative to the polyamine content after topping of an unmodifiedcontrol plant. In an aspect, a modified tobacco plant disclosedcomprises a modification conferring a desired trait (e.g., low-nicotine,nicotine-free, or low-alkaloid) and further comprises a polyaminecontent after topping which is between 70% and 140%, between 75% and135%, between 80% and 130%, between 85% and 125%, between 90% and 120%,between 95% and 115%, or between 100% and 110% relative to the polyaminecontent after topping of an unmodified control plant. In an aspect, amodified tobacco plant disclosed comprises a modification conferring adesired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) andfurther comprises a polyamine content after topping which is between 70%and 80%, between 75% and 85%, between 80% and 90%, between 85% and 95%,between 90% and 100%, between 95% and 105%, between 105% and 115%,between 110% and 120%, between 115% to 125%, between 120% and 130%,between 125 and 135%, or between 130% and 140% relative to the polyaminecontent after topping of an unmodified control plant.

In an aspect, a modified tobacco plant comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a chlorophyll level which is morethan 80%, more than 85%, more than 90%, more than 95%, more than 100%,more than 105%, more than 110%, more than 115%, more than 120%, morethan 125%, more than 130%, more than 135%, or more than 140% relative tothe chlorophyll level of an unmodified control plant. In an aspect, amodified tobacco plant disclosed comprises a modification conferring adesired trait (e.g., low-nicotine, nicotine-free, or low-alkaloid) andfurther comprises a chlorophyll level which is between 70% and 140%,between 75% and 135%, between 80% and 130%, between 85% and 125%,between 90% and 120%, between 95% and 115%, or between 100% and 110%relative to the chlorophyll level of an unmodified control plant. In anaspect, a modified tobacco plant disclosed comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a chlorophyll level which is between70% and 80%, between 75% and 85%, between 80% and 90%, between 85% and95%, between 90% and 100%, between 95% and 105%, between 105% and 115%,between 110% and 120%, between 115% to 125%, between 120% and 130%,between 125 and 135%, or between 130% and 140% relative to thechlorophyll level of an unmodified control plant.

In an aspect, a modified tobacco plant comprises a modificationconferring a desired trait (e.g., low-nicotine, nicotine-free, orlow-alkaloid) and further comprises a mesophyll cell number per unitleaf area which is more than 80%, more than 85%, more than 90%, morethan 95%, more than 100%, more than 105%, more than 110%, more than115%, more than 120%, more than 125%, more than 130%, more than 135%, ormore than 140% relative to the mesophyll cell number per unit leaf areaof an unmodified control plant. In an aspect, a modified tobacco plantdisclosed comprises a modification conferring a desired trait (e.g.,low-nicotine, nicotine-free, or low-alkaloid) and further comprises amesophyll cell number per unit leaf area which is between 70% and 140%,between 75% and 135%, between 80% and 130%, between 85% and 125%,between 90% and 120%, between 95% and 115%, or between 100% and 110%relative to the mesophyll cell number per unit leaf area of anunmodified control plant. In an aspect, a modified tobacco plantdisclosed comprises a modification conferring a desired trait (e.g.,low-nicotine, nicotine-free, or low-alkaloid) and further comprises amesophyll cell number per unit leaf area which is between 70% and 80%,between 75% and 85%, between 80% and 90%, between 85% and 95%, between90% and 100%, between 95% and 105%, between 105% and 115%, between 110%and 120%, between 115% to 125%, between 120% and 130%, between 125 and135%, or between 130% and 140% relative to the mesophyll cell number perunit leaf area of an unmodified control plant.

In an aspect, a low-nicotine or nicotine-free tobacco variety is adaptedfor machine harvesting. In another aspect, a low-nicotine ornicotine-free tobacco variety disclosed is harvested mechanically.

In an aspect, a method for improving leaf quality in a reduced-alkaloidtobacco plant is provided, the method comprising: growing a tobaccoplant; reducing the level of putrescine in the tobacco plant, andharvesting leaves from the tobacco plant.

In an aspect, a method for improving leaf quality in a reduced-alkaloidtobacco plant is provided, the method comprising: growing a tobaccoplant; suppressing the expression or activity of an ornithinedecarboxylase (ODC) gene in the tobacco plant, and harvesting leavesfrom the tobacco plant. In one aspect, the suppressing step is within 2,4, 6, or 8 WPT. In an aspect, the suppressing step comprises suppressinga ODC gene both prior to and after topping a tobacco plant. In oneaspect, the suppressing step does not include the use of a chemicalinhibitor. In an aspect, the suppressing step is accomplished byinducing the expression of a non-coding RNA for suppression of anornithine decarboxylase (ODC) gene. In one aspect, the suppressing stepcomprises applying an ODC inhibitor to the tobacco plant. In an aspect,the suppressing is accomplished by applying an ODC inhibitor to atobacco plant. In one aspect, an ODC inhibitor is DFMO.

In an aspect, tobacco plants provided are hybrid plants. Hybrids can beproduced by preventing self-pollination of female parent plants (e.g.,seed parents) of a first variety, permitting pollen from male parentplants of a second variety to fertilize the female parent plants, andallowing F1 hybrid seeds to form on the female plants. Self-pollinationof female plants can be prevented by emasculating the flowers at anearly stage of flower development. Alternatively, pollen formation canbe prevented on the female parent plants using a form of male sterility.For example, male sterility can be produced by male sterility (MS), ortransgenic male sterility where a transgene inhibits microsporogenesisand/or pollen formation, or self-incompatibility. Female parent plantscontaining MS are particularly useful. In aspects in which the femaleparent plants are MS, pollen may be harvested from male fertile plantsand applied manually to the stigmas of MS female parent plants, and theresulting F1 seed is harvested.

Plants can be used to form single-cross tobacco F1 hybrids. Pollen froma male parent plant is manually transferred to an emasculated femaleparent plant or a female parent plant that is male sterile to form F1seed. Alternatively, three-way crosses can be carried out where asingle-cross F1 hybrid is used as a female parent and is crossed with adifferent male parent. As another alternative, double-cross hybrids canbe created where the F1 progeny of two different single-crosses arethemselves crossed. Self-incompatibility can be used to particularadvantage to prevent self-pollination of female parents when forming adouble-cross hybrid.

In an aspect, a low-nicotine or nicotine-free tobacco variety is malesterile. In another aspect, a low-nicotine or nicotine-free tobaccovariety is cytoplasmic male sterile. Male sterile tobacco plants may beproduced by any method known in the art. Methods of producing malesterile tobacco are described in Wernsman, E. A., and Rufty, R. C. 1987.Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. CropSpecies. W. H. Fehr (ed.), MacMillan Publishing Go, Inc., New York, N.Y.761 pp.

In a further aspect, tobacco parts provided include, but are not limitedto, a leaf, a stem, a root, a seed, a flower, pollen, an anther, anovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod,an embryo, endosperm, an explant, a callus, a tissue culture, a shoot, acell, and a protoplast. In an aspect, tobacco part provided does notinclude seed. In an aspect, this disclosure provides tobacco plantcells, tissues, and organs that are not reproductive material and do notmediate the natural reproduction of the plant. In another aspect, thisdisclosure also provides tobacco plant cells, tissues, and organs thatare reproductive material and mediate the natural reproduction of theplant. In another aspect, this disclosure provides tobacco plant cells,tissues, and organs that cannot maintain themselves via photosynthesis.In another aspect, this disclosure provides somatic tobacco plant cells.Somatic cells, contrary to germline cells, do not mediate plantreproduction.

The provided cells, tissues and organs may be from seed, fruit, leaf,cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem,pod, flower, inflorescence, stalk, pedicel, style, stigma, receptacle,petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem,vascular tissue. In another aspect, this disclosure provides a tobaccoplant chloroplast. In a further aspect, this disclosure providesepidermal cells, stomata cell, leaf or root hairs, a storage root, or atuber. In another aspect, this disclosure provides a tobacco protoplast.

Skilled artisans understand that tobacco plants naturally reproduce viaseeds, not via asexual reproduction or vegetative propagation. In anaspect, this disclosure provides tobacco endosperm. In another aspect,this disclosure provides tobacco endosperm cells. In a further aspect,this disclosure provides a male or female sterile tobacco plant, whichcannot reproduce without human intervention.

In an aspect, the present disclosure provides a nucleic acid moleculecomprising at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto a sequence selected from the group consisting of SEQ ID NOs: 23-28,and fragments thereof. In an aspect, the present disclosure provides apolypeptide or protein comprising at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 29-34. In another aspect, the presentdisclosure provides a biologically active variant of a protein having anamino acid sequence selected from the group consisting of SEQ ID NOs:29-34. A biologically active variant of a protein of the presentdisclosure may differ from that protein by as few as 1-15 amino acidresidues, as few as 10, as few as 9, as few as 8, as few as 7, as few as6, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1amino acid residue. Also provided are orthologous genes or proteins ofgenes or proteins from the ODC pathway. “Orthologs” are genes derivedfrom a common ancestral gene and which are found in different species asa result of speciation. Orthologs may share at least 60%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greatersequence identity or similarity at the nucleotide sequence and/or theprotein sequence level. Functions of orthologs are often highlyconserved among species.

As used herein, the term “sequence identity” or “identity” in thecontext of two polynucleotides or polypeptide sequences makes referenceto the residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare deemed to have “sequence similarity” or “similarity.”

Nucleic acid molecules, polypeptides, or proteins provided can beisolated or substantially purified. An “isolated” or “purified” nucleicacid molecule, polypeptide, protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the polynucleotide or protein asfound in its naturally occurring environment. For example, an isolatedor purified polynucleotide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The present disclosure further provides a method manufacturing a tobaccoproduct comprising tobacco material from tobacco plants disclosed. In anaspect, methods comprise conditioning aged tobacco material made fromtobacco plants to increase its moisture content from between about 12.5%and about 13.5% to about 21%, blending the conditioned tobacco materialto produce a desirable blend. In an aspect, the method of manufacturinga tobacco product further comprises casing or flavoring the blend.Generally, during the casing process, casing or sauce materials areadded to blends to enhance their quality by balancing the chemicalcomposition and to develop certain desired flavor characteristics.Further details for the casing process can be found in TobaccoProduction, Chemistry and Technology, Edited by L. Davis and M. Nielsen,Blackwell Science, 1999.

Tobacco material provided can be also processed using methods including,but not limited to, heat treatment (e.g., cooking, toasting), flavoring,enzyme treatment, expansion and/or curing. Both fermented andnon-fermented tobaccos can be processed using these techniques. Examplesof suitable processed tobaccos include dark air-cured, dark fire cured,burley, flue cured, and cigar filler or wrapper, as well as the productsfrom the whole leaf stemming operation. In an aspect, tobacco fibersinclude up to 70% dark tobacco on a fresh weight basis. For example,tobacco can be conditioned by heating, sweating and/or pasteurizingsteps as described in U.S. Publication Nos. 2004/0118422 or2005/0178398.

Tobacco material provided can be subject to fermentation. Fermentingtypically is characterized by high initial moisture content, heatgeneration, and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat.Nos. 4,528,993; 4,660,577; 4,848,373; and 5,372,149. In addition tomodifying the aroma of the leaf, fermentation can change either or boththe color and texture of a leaf. Also during the fermentation process,evolution gases can be produced, oxygen can be taken up, the pH canchange, and the amount of water retained can change. See, for example,U.S. Publication No. 2005/0178398 and Tso (1999, Chapter 1 in Tobacco,Production, Chemistry and Technology, Davis & Nielsen, eds., BlackwellPublishing, Oxford). Cured, or cured and fermented tobacco can befurther processed (e.g., cut, expanded, blended, milled or comminuted)prior to incorporation into the oral product. The tobacco, in somecases, is long cut fermented cured moist tobacco having an ovenvolatiles content of between 48 and 50 weight percent prior to mixingwith the copolymer and optionally flavorants and other additives.

In an aspect, tobacco material provided can be processed to a desiredsize. In an aspect, tobacco fibers can be processed to have an averagefiber size of less than 200 micrometers. In an aspect, tobacco fibersare between 75 and 125 micrometers. In another aspect, tobacco fibersare processed to have a size of 75 micrometers or less. In an aspect,tobacco fibers include long cut tobacco, which can be cut or shreddedinto widths of about 10 cuts/inch up to about 110 cuts/inch and lengthsof about 0.1 inches up to about 1 inch. Double cut tobacco fibers canhave a range of particle sizes such that about 70% of the double cuttobacco fibers falls between the mesh sizes of −20 mesh and 80 mesh.

Tobacco material provided can be processed to have a total ovenvolatiles content of about 10% by weight or greater; about 20% by weightor greater; about 40% by weight or greater; about 15% by weight to about25% by weight; about 20% by weight to about 30% by weight; about 30% byweight to about 50% by weight; about 45% by weight to about 65% byweight; or about 50% by weight to about 60% by weight. Those of skill inthe art will appreciate that “moist” tobacco typically refers to tobaccothat has an oven volatiles content of between about 40% by weight andabout 60% by weight (e.g., about 45% by weight to about 55% by weight,or about 50% by weight). As used herein, “oven volatiles” are determinedby calculating the percentage of weight loss for a sample after dryingthe sample in a pre-warmed forced draft oven at 110° C. for 3.25 hours.The oral product can have a different overall oven volatiles contentthan the oven volatiles content of the tobacco fibers used to make theoral product. The processing steps described can reduce or increase theoven volatiles content.

Having now generally described the disclosure, the same will be morereadily understood through reference to the following examples that areprovided by way of illustration, and are not intended to be limiting ofthe present disclosure, unless specified.

The following are exemplary embodiments of the present disclosure.

Embodiment 1. A tobacco plant comprising an inducible promoter operablylinked to a transcribable DNA sequence encoding a non-coding RNA forsuppression of an ornithine decarboxylase (ODC) gene.

Embodiment 2. The tobacco plant of Embodiment 1, wherein said tobaccoplant comprises a mutation or a transgene conferring a reduced level ofnicotine.

Embodiment 3. The tobacco plant of Embodiments 1 or 2, wherein saidtobacco plant is a low-alkaloid tobacco plant.

Embodiment 4. The tobacco plant of any one of Embodiments 1-3, whereinsaid tobacco plant comprises a nic1 mutation, a nic2 mutation, or both.

Embodiment 5. The tobacco plant of any one of Embodiments 1-4, whereinsaid tobacco plant comprises a mutation in a gene or locus encoding aprotein selected from the group consisting of aspartate oxidase,agmatine deiminase (AIC), arginase, diamine oxidase, argininedecarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase,ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase(PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1,BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcriptionfactor, nicotine uptake permease (NUP), and MATE transporter.

Embodiment 6. The tobacco plant of any one of Embodiments 1-5, whereinsaid tobacco plant comprises a mutation in a gene or locus encoding aprotein selected from the group consisting of ERF32, ERF34, ERF39,ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

Embodiment 7. The tobacco plant of any one of Embodiments 1-6, whereinsaid tobacco plant comprises a transgene targeting and suppressing agene encoding a protein selected from the group consisting of aspartateoxidase, agmatine deiminase (AIC), arginase, diamine oxidase, argininedecarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase,ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase(PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1,BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF) transcriptionfactor, nicotine uptake permease (NUP), and MATE transporter.

Embodiment 8. The tobacco plant of any one of Embodiments 1-7, whereinsaid tobacco plant comprises a transgene targeting and suppressing agene encoding a protein selected from the group consisting of ERF32,ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

Embodiment 9. The tobacco plant of any one of Embodiments 1-8, whereinsaid tobacco plant is capable of producing a leaf comprising acomparable level of one or more polyamines relative to a comparable leafof a control plant not comprising said mutation or said transgene.

Embodiment 10. The tobacco plant of any one of Embodiments 1-9, whereinsaid comparable level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%,2.5%, or 1% of the level in said control.

Embodiment 11. The tobacco plant of any one of claims Embodiments 1-10,wherein said tobacco plant is capable of producing a leaf comprising acomparable chlorophyll level relative to a comparable leaf of a controlplant not comprising said mutation or said transgene.

Embodiment 12. The tobacco plant of any one of Embodiments 1-11, whereinsaid comparable chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%,7.5%, 5%, 2.5%, or 1% of the level in said control.

Embodiment 13. The tobacco plant of any one of Embodiments 1-12, whereinsaid tobacco plant is capable of producing a leaf comprising acomparable number of mesophyll cell per unit of leaf area relative to acomparable leaf of a control plant not comprising said mutation or saidtransgene.

Embodiment 14. The tobacco plant of any one of Embodiments 1-13, whereinsaid comparable mesophyll cell per unit of leaf area is within 20%,17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in saidcontrol.

Embodiment 15. The tobacco plant of any one of Embodiments 1-14, whereinsaid tobacco plant is capable of producing a leaf comprising acomparable epidermal cell size relative to a comparable leaf of acontrol plant not comprising said mutation or said transgene.

Embodiment 16. The tobacco plant of any one of Embodiments 1-15, whereinsaid comparable epidermal cell size is within 20%, 17.5%, 15%, 12.5%,10%, 7.5%, 5%, 2.5%, or 1% of the level in said control.

Embodiment 17. The tobacco plant of any one of Embodiments 1-16, whereinsaid tobacco plant comprises a comparable leaf yield relative to acomparable leaf of a control plant not comprising said mutation or saidtransgene.

Embodiment 18. The tobacco plant of any one of Embodiments 1-17, whereinsaid comparable leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%,5%, 2.5%, or 1% of the level in said control.

Embodiment 19. The tobacco plant of any one of Embodiments 1-18, whereinsaid tobacco plant exhibits a comparable insect herbivory susceptibilityrelative to a comparable leaf of a control plant not comprising saidmutation or said transgene.

Embodiment 20. The tobacco plant of any one of Embodiments 1-19, whereinsaid ornithine decarboxylase (ODC) gene encodes a polypeptide sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99%, or 100% identity to a sequence selected from thegroup consisting of SEQ ID Nos: 29-34.

Embodiment 21. The tobacco plant of any one of Embodiments 1-20, whereinsaid ODC gene comprises a nucleotide sequence having at least 90%, atleast 95%, at least 97%, at least 99%, or 100% identity to a sequenceselected from the group consisting of SEQ ID Nos: 23-28.

Embodiment 22. The tobacco plant of any one of Embodiments 1-21, whereinsaid inducible promoter is a topping-inducible promoter.

Embodiment 23. The tobacco plant of any one of Embodiments 1-22, whereinsaid inducible promoter is also a tissue-specific or tissue-preferredpromoter.

Embodiment 24. The tobacco plant of any one of Embodiments 1-23, whereinsaid tissue-specific or tissue-preferred promoter is specific orpreferred for one or more tissues or organs selected from the groupconsisting of shoot, root, leaf, stem, flower, sucker, root tip,mesophyll cells, epidermal cells, and vasculature.

Embodiment 25. The tobacco plant of any one of Embodiments 1-24, whereinsaid inducible promoter regulates root specific or preferred expression.

Embodiment 26. The tobacco plant of any one of Embodiments 1-25, whereinsaid inducible promoter comprises a sequence selected from the groupconsisting of SEQ ID Nos: 1-11.

Embodiment 27. The tobacco plant of any one of Embodiments 1-26, whereinsaid inducible promoter regulates leaf specific or preferred expression.

Embodiment 28. The tobacco plant of any one of Embodiments 1-27, whereinsaid inducible promoter comprises a sequence selected from the groupconsisting of SEQ ID Nos: 12-21.

Embodiment 29. The tobacco plant of any one of Embodiments 1-28, whereinsaid inducible promoter is a heterologous to said transcribable DNAsequence.

Embodiment 30. The tobacco plant of any one of Embodiments 1-29, whereinsaid non-coding RNA is selected from the group consisting of microRNA(miRNA), anti-sense RNA, small interfering RNA (siRNA), a trans-actingsiRNA (ta-siRNA), and hairpin RNA (hpRNA).

Embodiment 31. The tobacco plant of any one of Embodiments 1-30, whereinsaid non-coding RNA comprises a nucleotide sequence having at least 90%,at least 95%, at least 97%, at least 99%, or 100% identity to a sequenceselected from the group consisting of SEQ ID Nos: 35 and 36.

Embodiment 32. The tobacco plant of any one of Embodiments 1-31, whereinsaid non-coding RNA is provided in an ODC RNAi construct comprising anucleotide sequence having at least 90% identity to SEQ ID No: 22.

Embodiment 33. A tobacco plant, or part thereof, comprising a nic1mutation, a nic2 mutation, or both, and further comprising a transgeneor mutation providing an early-senescence trait.

Embodiment 34. The tobacco plant of Embodiment 33, wherein said mutationproviding an early-senescence trait is yellow burley1 (−yb1).

Embodiment 35. The tobacco plant of Embodiments 33 or 34, wherein saidmutation providing an early-senescence trait is yellow burley2 (−yb2).

Embodiment 36. The tobacco plant of any one of Embodiments 33-35,wherein said mutation providing an early-senescence trait is pale yellow(PY).

Embodiment 37. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        one or more traits selected from the group consisting of        -   i. total leaf polyamine level,        -   ii. total root polyamine level,        -   iii. total leaf chlorophyll level,        -   iv. mesophyll cell number per leaf area unit, and        -   v. leaf epidermal cell size; and    -   wherein said control plant does not have both said first and        said second genome modifications.

Embodiment 38. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        total leaf polyamine level, wherein said control plant does not        have both said first and said second genome modifications.

Embodiment 39. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        total root polyamine level, wherein said control plant does not        have both said first and said second genome modifications.

Embodiment 40. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        total leaf chlorophyll level, wherein said control plant does        not have both said first and said second genome modifications.

Embodiment 41. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        mesophyll cell number per leaf area unit, wherein said control        plant does not have both said first and said second genome        modifications.

Embodiment 42. A tobacco plant, or part thereof, comprising relative toa control tobacco plant:

-   -   a. a first genome modification providing a lower level of        nicotine or total alkaloid, and    -   b. a second genome modification providing a comparable level of        leaf epidermal cell size, wherein said control plant does not        have both said first and said second genome modifications.

Embodiment 43. The tobacco plant, or part thereof, of any one ofEmbodiments 37-42, wherein said tobacco plant comprises a reduced amountof total conjugated polyamines in leaves relative to said controltobacco plant.

Embodiment 44. The tobacco plant, or part thereof, of any one ofEmbodiments 37-43, wherein said tobacco plant comprises a reduced amountof total conjugated polyamines in roots relative to said control tobaccoplant.

Embodiment 45. The tobacco plant, or part thereof, of any one ofEmbodiments 37-44, wherein said tobacco plant comprises a reduced amountof total free polyamines in leaves relative to said control tobaccoplant.

Embodiment 46. The tobacco plant, or part thereof, of any one ofEmbodiments 37-45, wherein said tobacco plant comprises a reduced amountof total conjugated polyamines in roots relative to said control tobaccoplant.

Embodiment 47. The tobacco plant, or part thereof, of any one ofEmbodiments 37-46, wherein said tobacco plant comprises a reduced amountof total conjugated form of one or more polyamines selected from thegroup consisting of putrescine, spermidine and spermine in leavesrelative to said control tobacco plant.

Embodiment 48. The tobacco plant, or part thereof, of any one ofEmbodiments 37-47, wherein said tobacco plant comprises a reduced amountof total conjugated form of one or more polyamines selected from thegroup consisting of putrescine, spermidine and spermine in rootsrelative to said control tobacco plant.

Embodiment 49. The tobacco plant, or part thereof, of any one ofEmbodiments 37-48, wherein said tobacco plant comprises a reduced amountof total free form of one or more polyamines selected from the groupconsisting of putrescine, spermidine and spermine in leaves relative tosaid control tobacco plant.

Embodiment 50. The tobacco plant, or part thereof, of any one ofEmbodiments 37-49, wherein said tobacco plant comprises a reduced amountof total conjugated form of one or more polyamines selected from thegroup consisting of putrescine, spermidine and spermine in rootsrelative to said control tobacco plant.

Embodiment 51. The tobacco plant, or part thereof, of any one ofEmbodiments 37-50, wherein said first genome modification provides alower level of nicotine compared to said control tobacco plant.

Embodiment 52. The tobacco plant, or part thereof, of any one ofEmbodiments 37-51, said first genome modification, said second genomemodification, or both comprise a transgene, a mutation, or both.

Embodiment 53. The tobacco plant, or part thereof, of any one ofEmbodiments 37-52, said first genome modification, said second genomemodification, or both comprise a transgene.

Embodiment 54. The tobacco plant, or part thereof, of any one ofEmbodiments 37-53, said first genome modification, said second genomemodification, or both comprise a mutation.

Embodiment 55. The tobacco plant, or part thereof, of any one ofEmbodiments 37-54, said first genome modification, said second genomemodification, or both are not transgene-based.

Embodiment 56. The tobacco plant, or part thereof, of any one ofEmbodiments 37-55, said first genome modification, said second genomemodification, or both are not mutation-based.

Embodiment 57. The tobacco plant, or part thereof, of any one ofEmbodiments 37-56, wherein said first genome modification comprises anic1 mutation, a nic2 mutation, or both.

Embodiment 58. The tobacco plant, or part thereof, of any one ofEmbodiments 37-57, wherein said first genome modification comprises atransgene targeting the Nic1 locus, a transgene targeting the Nic2locus, or both.

Embodiment 59. The tobacco plant, or part thereof, of any one ofEmbodiments 37-58, wherein said second genome modification comprises atranscribable DNA sequence encoding a non-coding RNA for suppression ofan ornithine decarboxylase (ODC) gene, a MYB8 gene, or both.

Embodiment 60. The tobacco plant, or part thereof, of any one ofEmbodiments 37-59, wherein said a transcribable DNA sequence is operablylinked to an heterologous promoter selected from the group consisting ofa constitutive promoter, a developmental promoter, a tissue-specificpromoter, a tissue-preferred promoter, an inducible promoter, and anycombination thereof.

Embodiment 61. The tobacco plant, or part thereof, of any one ofEmbodiments 37-60, wherein said second genome modification comprisesoverexpression of an diamine oxidase, suppression of an argininedecarboxylase, or both.

Embodiment 62. The tobacco plant, or part thereof, of any one ofEmbodiments 37-61, wherein said first genome modification comprises amutation in a gene or locus encoding a protein selected from the groupconsisting of aspartate oxidase, agmatine deiminase (AIC), arginase,diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase(MPO), NADH dehydrogenase, ornithine decarboxylase (ODC),phosphoribosylanthranilate isomerase (PRAI), putrescineN-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT),and S-adenosyl-methionine synthetase (SAMS), A622, NBB1, BBL, MYC2,Nic1, Nic2, ethylene response factor (ERF) transcription factor,nicotine uptake permease (NUP), and MATE transporter.

Embodiment 63. The tobacco plant, or part thereof, of any one ofEmbodiments 37-62, wherein said first genome modification comprises amutation in a gene or locus encoding a protein selected from the groupconsisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104,ERF179, ERF17, and ERF168.

Embodiment 64. The tobacco plant, or part thereof, of any one ofEmbodiments 37-63, wherein said first genome modification comprises atransgene targeting and suppressing a gene or locus encoding a proteinselected from the group consisting of aspartate oxidase, agmatinedeiminase (AIC), arginase, diamine oxidase, arginine decarboxylase(ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithinedecarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),putrescine N-methyltransferase (PMT), quinolate phosphoribosyltransferase (QPT), and S-adenosyl-methionine synthetase (SAMS), A622,NBB1, BBL, MYC2, Nic1, Nic2, ethylene response factor (ERF)transcription factor, nicotine uptake permease (NUP), and MATEtransporter.

Embodiment 65. The tobacco plant, or part thereof, of any one ofEmbodiments 37-64, wherein said first genome modification comprises atransgene targeting and suppressing a gene or locus encoding a proteinselected from the group consisting of ERF32, ERF34, ERF39, ERF189,ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.

Embodiment 66. The tobacco plant, or part thereof, of any one ofEmbodiments 37-65, wherein said lower level is measured at a timeselected from the group consisting of immediately before flowering, attopping, 1 week-post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7WPT, 8 WPT, and at harvest.

Embodiment 67. The tobacco plant, or part thereof, of any one ofEmbodiments 37-66, wherein said comparable level is measured at a timeselected from the group consisting of immediately before flowering, attopping, 1 week-post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7WPT, 8 WPT, and at harvest.

Embodiment 68. The tobacco plant, or part thereof, of any one ofEmbodiments 37-67, wherein said tobacco plant is capable of producing aleaf with a leaf grade comparable to that of a leaf from said controlplant.

Embodiment 69. The tobacco plant, or part thereof, of any one ofEmbodiments 37-68, wherein said tobacco plant has a total leaf yieldcomparable to said control plant.

Embodiment 70. The tobacco plant, or part thereof, of any one of thepreceding Embodiments, wherein said tobacco plant comprises a nicotinelevel selected from the group consisting of less than 3%, less than2.75%, less than 2.5%, less than 2.25%, less than 2.0%, less than 1.75%,less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%,less than 0.3%, less than 0.2%, less than 0.1%, and less than 0.05%.

Embodiment 71. The tobacco plant, or part thereof, of any one ofEmbodiments 37-70, wherein said tobacco plant comprises nicotine at alevel below 1%, below 2%, below 5%, below 8%, below 10%, below 12%,below 15%, below 20%, below 25%, below 30%, below 40%, below 50%, below60%, below 70%, or below 80% of the nicotine level of said control plantwhen grown in comparable growth conditions.

Embodiment 72. A population of the tobacco plants of any one of thepreceding Embodiments.

Embodiment 73. Cured tobacco material from the tobacco plant of any oneof the preceding Embodiments.

Embodiment 74. The cured tobacco material of Embodiment 73, wherein saidcured tobacco material is made by a curing process selected from thegroup consisting of flue curing, air curing, fire curing, and suncuring.

Embodiment 75. A tobacco blend comprising said cured tobacco material ofEmbodiments 73 or 74.

Embodiment 76. The tobacco blend of any one of Embodiments 73-75,wherein said cured tobacco material constitutes about at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% of cured tobacco in said tobacco blend byweight.

Embodiment 77. The tobacco blend of any one of Embodiments 73-76,wherein said cured tobacco material constitutes about at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% of cured tobacco in said tobacco blend byvolume.

Embodiment 78. A tobacco product comprising the cured tobacco materialof any one of Embodiments 73-77.

Embodiment 79. The tobacco product of Embodiment 78, wherein saidtobacco product is selected from the group consisting of a cigarette, acigarillo, a non-ventilated recess filter cigarette, a vented recessfilter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarettetobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cuttobacco.

Embodiment 80. The tobacco product of Embodiments 78 or 79, wherein saidtobacco product is selected from the group consisting of loose leafchewing tobacco, plug chewing tobacco, moist snuff, and nasal snuff.

Embodiment 81. A method for improving leaf quality in a reduced-alkaloidtobacco plant, said method comprising:

-   -   a. Growing a tobacco plant;    -   b. Reducing the level of putrescine in said tobacco plant,    -   c. Harvesting leaves from said tobacco plant.

Embodiment 82. A method for improving leaf quality in a reduced-alkaloidtobacco plant, said method comprising:

-   -   a. Growing a tobacco plant;    -   b. Suppressing the expression or activity of an ornithine        decarboxylase (ODC) gene in said tobacco plant,    -   c. Harvesting leaves from said tobacco plant.

Embodiment 83. The method of Embodiment 81 or 82, wherein saidsuppressing is within 2, 4, 6, or 8 WPT.

Embodiment 84. The method of any one of Embodiments 81-83, wherein saidsuppressing comprises suppressing said ODC gene both prior to and aftertopping said tobacco plant.

Embodiment 85. The method of any one of Embodiments 82-84, wherein saidsuppressing does not include the use of a chemical inhibitor.

Embodiment 86. The method of any one of Embodiments 82-85, wherein saidsuppressing comprises applying an ODC inhibitor to said tobacco plant.

Embodiment 87. The method of any one of Embodiments 82-86, wherein saidsuppressing is by applying an ODC inhibitor to said tobacco plant.

Embodiment 88. The method of any one of Embodiments 82-87, wherein saidODC inhibitor is DFMO.

Embodiment 89. The method of any one of Embodiments 82-88, wherein saidsuppressing is by inducing the expression of a non-coding RNA forsuppression of said ornithine decarboxylase (ODC) gene.

Embodiment 90. The method of any one of Embodiments 82-89, wherein saidmethod further comprises reducing nitrogen fertilization or reducingnitrate.

EXAMPLES Example 1: Plant Material and General Growth Conditions

Seeds of Nicotiana tabacum L. cv. Burley 21 wild-type NA, as well as HI(nic2), LI (nic1) and LA (nic1nic2) near-isogenic varieties wereobtained from the US Nicotiana Germplasm Collection at North CarolinaState University and used in all greenhouse experiments. Seeds weregerminated in pots under greenhouse conditions at 27/23° C. day/nighttemperature and a 16-h photoperiod (˜200 mmol s⁻¹ m⁻²; λ=400-700 nm) at70% relative humidity. Five week-old tobacco plantlets were transferredto 13-L pots with standard substrate (Einheitserde, Fröndenberg,Germany). The plants were attached to a continuous drip irrigationsystem active every 4 h for ˜5 min and were irrigated with 0.7% (w/v)Ferty 2 Mega containing 16% nitrogen, (Planta Düngemittel, Regenstauf,Germany) for 5 min h⁻¹ during the 16-h photoperiod and grown for 4additional weeks. Greenhouse plants were topped when 50% of the plantshad at least one open flower. After topping, plants grew for additional4-7 weeks until harvest, 30 days post-topping. Tobacco leaves used forpolyamine analysis were collected from greenhouse-grown plants at threetime points: before flowering (6.5-week-old plants), just beforetopping, i.e. the removal of the floral apex (9-week-old plants) and atharvest (13-week-old plants, 4 weeks post-topping). Root samples werecollected at topping and harvest. LA and NA tobacco plants were grown inthe field under 135 units of nitrogen per acre and sampled at 1 weekpost-topping for polyamine analysis.

For treatment with polyamine biosynthesis inhibitors, 5 mM D-Arginine(AKos, Steinen, Germany), 2 mM DFMO (Synchem Ug & Co. KG, Felsberg,Germany) alone or in combination with 0.5 mM Ethephon®, or 0.5 mMEthephon® alone (Merck KGaA, Darmstadt, Germany), were diluted in thesame amount of water used for daily irrigation and applied to LA plantsevery 4 hours at 9 am, 12 am, 3 pm and 6 pm three times per week insteadof the drip irrigation system. The treatment with D-arginine and DFMOstarted before flowering (˜2.5 weeks before topping when plants werestill in the vegetative growth stage) for a period of 6 weeks untilharvest, whereas Ethephon® was applied from topping to harvest (4 weeksin total) to avoid early senescence in the LA plants. Twelve plants perinhibitor were treated. NA plants were used as controls and treated inthe same way as the LA plants. More description of experimentalprocedures and data can be found at Nölke G, et al. Polyamines delayleaf maturation in low-alkaloid tobacco varieties. Plant Direct. 2018;2:1-12.

Example 2: Chlorophyll Measurements

Chlorophyll contents were determined by measuring leaf absorbance in thered and infrared regions using a SPAD-502 Plus device (Minolta CameraCo., Osaka, Japan). Chlorophyll was measured twice in the same day atdifferent positions in all fully expanded (length>15 cm) leaves (leaves6-26) from six randomly-selected plants from each line at five growthstages: before flowering (6.5-week-old plants and 2.5 weeks beforetopping), at topping, 1 and 2.5 WPT and at harvest (30 days posttopping) before flowering. The total chlorophyll content was calculatedas an average of all measured leaf chlorophyll values per plant tominimize the influence of leaf position.

Example 3: Leaf Cell Microscopy

Four leaf discs (1 cm²) cut form leaf 15 from six biological replicatesat different development stages (before flowering, at topping, 1 WPT andat harvest) were mounted on slides and imaged using a Leica DM Rmicroscope (Leica, Wetzlar, Germany) with a 10× air objective. Imageswere imported into ImageJ and Adobe Photoshop CS5.1 software and thecells per unit area were counted using Count Tool in the Photoshop CS5.A standard area was designated to use for cell counting three timesacross all images and care was taken to avoid counting any cell twice.

Example 4: Determination of ODC and ADC Activities

To determine enzymatic activities, 500 mg of tobacco leaf tissuecollected from leaf 23 of three biological replicates of was ground in 1ml HEPES extraction buffer (100 mM HEPES, 2 mM dithiothreitol (DTT), 1mM EDTA, pH 7.5) and 100 mg of polyvinylpyrrolidone was added duringgrinding. Following centrifugation (13,000 g, 10 min, 4° C.), the enzymeactivities were measured using an isotopic method as described by Capellet al. (1998) by measuring the release of ¹⁴CO2. L-[1-¹⁴C]Arg andL-[1-¹⁴C]Orn were used as radioactive substrates.

Example 5: Polyamine Extraction and Analysis

For polyamine analysis, 150 μg of leaf or root material was harvestedfrom plants grown in the greenhouse at different stages of development:before flowering (leaves 6 and 12, numbered from base), at topping(leaves 19 and 23 and roots) and at harvest (leaves 23 and 24 androots). Samples were collected after 4 h of illumination from threebiological replicates and were flash frozen in liquid nitrogen. Forfield-grown plants, leaf material was collected from five well-expandedupper leaves from three biological replicates. Plant material was groundin 1.6 ml pre-chilled 10% (v/v) perchloric acid and incubated at 4° C.for 1 h. The extract was vortexed for 10 s and centrifuged (16,000 g, 15min, 4° C.) before 800 μl of the supernatant was mixed with 100 μl 1 mMhexamethylenediamine. Then, 10 μl of the clear supernatant wastransferred to a fresh 2-ml tube and polyamines were extracted with 200μl of cyclohexane for the dansilation of free polyamines. For theextraction of conjugated polyamines, the pellet was resuspended in 1600μl 1 M NaOH and 200 μl 1 mM hexamethylenediamine and centrifuged asabove. The clear supernatant (200 μl) was transferred to a 2-ml glassampule containing 12 M HCl, mixed and incubated for 16 h at 110° C.overnight for the hydrolysis of conjugated polyamines. The dansilationof free and conjugated polyamines was carried out with dansyl chlorideas described by Flores and Galston (1982).

The dansylated polyamines were measured by LC-MS/MS. All experimentswere carried out on a 3200 QTRAP™ mass spectrometer (Sciex, Darmstadt,Germany) coupled to an HPLC Agilent 1200 system (Waldbronn, Germany).The mass spectrometer was equipped with an electrospray ionizationsource. The sample was separated on a reversed-phase Synergi Fusion with80 Å pore size, 4 μm particle size and dimensions of 50 mm×2.0 mminternal diameter (Phenomenex, Aschaffenburg, Germany) with thecorresponding guard column at a flow rate of 800 μl/min. The column ovenwas heated to 30° C. For elution, solvent A comprised 94.9% (v/v) water,5% (v/v) acetonitrile, 0.1% (v/v) formic acid and solvent B comprised94.9% (v/v) acetonitrile, 5% (v/v) water, 0.1% (v/v) formic acid. Theelution following elution profiles was used: 1 min, hold at 60% solventA/40% solvent B; 3 min, linear increase to 100% solvent B, 3 min hold at100% solvent B; rapid linear decrease to 60% solvent A/40% solvent B in0.1 min; hold for 1 min. The total run time was 8 min and the samplevolume injected in each run was 10 μl.

The mass spectrometer was set to unit resolution in Q1 and Q3. Allmeasurements were captured in multiple reaction monitoring mode. Forcompound optimization, standards were prepared according to thedansylation protocol, diluted in 50:50 (v/v) methanol/water and infusedwith a flow rate of 10 μl/min with the syringe pump directly connectedto the ion source. Declustering potential, collision energy, collisioncell entrance potential, collision cell exit potential and entrancepotential were optimized for all compounds using automated compoundoptimization (Table 3). The ion source parameters were set to: capillaryvoltage=5.5 kV, heater gas temperature=500° C., curtain gas=30 psi,nebulizing gas=70 psi, drying gas=70 psi, and collision gas=medium. Foreach analyte, one transition was used for quantification and another asa qualifier. The acquired data was processed using Analyst v1.6 (Sciex).The mass calibration of the 3200 QTRAP was achieved using polypropyleneglycol standards (Standards Chemical Kit with Low/High ConcentrationPPGs, Sciex) according to the manufacturer's instructions.

TABLE 3 Compound parameter for polyamine quantification. DP =declustering potential, CE = collision energy, CEP = collision cellentrance potential, CXP = collision cell exit potential, EP = entrancepotential. Dansylated Quantifier/ Parent Product DP EP CEP CE CXP RTCompound Qualifier mass [m/z] mass [m/z] [eV] [eV] [eV] [eV] [eV] [min]Spermine Quantifier 1135.39 360.3 86 10 48 65 4 4.3 Spermine Qualifier1135.39 170.3 86 10 48 121 4 4.3 Spermidine Quantifier 845.228 360.3 969.5 34 53 4 3.9 Spermidine Qualifier 845.228 170.3 96 9.5 34 81 4 3.9Putrescine Quantifier 555.119 170.3 61 7.5 24 45 4 3.2 PutrescineQualifier 555.119 168.3 61 7.5 24 79 4 3.2 Hexamethyldiamine Quantifier583.14 170.3 70 10 28.809 50 4 3.5 Hexamethyldiamine Qualifier 583.14169.2 70 10 28.809 50 4 3.5

Example 6: Statistical Analysis

Differences between the genotypes were determined by applying one-wayanalysis of variance (ANOVA) followed by post-hoc Bonferroni test usingExcel software (Microsoft, Redmond, Wash., USA). Two-tailed t-tests wereapplied. A p-value<0.05 was considered statistically significant.

Example 7: Biochemical and Morphological Differences Among the FourVarieties During Leaf Ripening

Progression of senescence in the Burley 21 NA, HI, LI and LA lines wasmonitored by measuring the loss of chlorophyll a and b in the leaves.The chlorophyll levels had declined significantly (p<0.01) in allgenotypes after 1 week post-topping (WPT) (FIG. 1A). However, the leavesof the LI and LA plants contained significantly (p<0.001) higher levelsof chlorophyll than the NA controls at 2.5 WPT (22% more in bothgenotypes) and at harvest (36% and 44% more in the LI and LA leaves,respectively), indicating slower chlorophyll degradation compared to NAcontrols. Loss of chlorophyll was correlated with morphological changesin the leaves of NA plants, i.e. they became wrinkly and leathery withyellow patches, whereas the LA leaves remained smooth, shiny and green(FIG. 1B).

Given the distinct leaf morphology in the LA and NA lines, the size andshape of the mesophyll cells were investigated at different time points.Before flowering, leaf 15 (numbered from the base) of the LA plants hadsmaller and more abundant mesophyll cells (more cells per unit leafarea) compared to the NA plants (FIG. 1C/D). From that time point untilharvest, the number of leaf mesophyll cells per unit area declined at asimilar rate in both the NA and LA lines, but the LA plants retained asignificantly (p<0.05) greater number of mesophyll cells throughoutripening. The greatest difference in mesophyll cell number per unit area(54% more cells in the LA plants compared to NA controls) was observedat earlier stages of leaf development (before flowering). LI plants alsocontained more mesophyll cells than the NA plants but not to the degreeobserved in the LA plants, and there was no significant difference inmesophyll cell number between the HI and NA lines (data not shown).

Example 8: LA Plants Accumulate Higher Levels of Polyamines than NAPlants

To investigate the impact of the nic1nic2 double mutation on polyaminebiosynthesis, the levels of free and conjugated putrescine, spermidineand spermine in the NA and LA plants were compared by liquidchromatography tandem mass spectrometry (LC-MS/MS). First, the polyaminecontent were analyzed in leaves 16-18 of field-grown plants. At 1 WPT,the total polyamine content was significantly higher (1.9-fold, p<0.001)in the LA plants compared to the NA plants (FIG. 2A). Compositionalanalysis revealed significantly higher levels of free putrescine(1.4-fold, p<0.05), conjugated putrescine (2.3-fold, p<0.005) andconjugated spermidine (1.9-fold, (p<0.005) levels in the leaves of theLA plants, indicating that the polyamine biosynthesis pathway isstrongly induced by the nic1nic2 double mutant or that the inability ofthe substrates to be further processed into nicotine results in abuildup of these materials. In contrast, the level of free spermidine inthe LA plants was lower than in the NA plants, although the differencewas not statistically significant (p>0.05).

To minimize the effect of variable environmental factors on polyaminebiosynthesis, further experiments were performed under controlledgreenhouse conditions mirroring the average field conditions in terms oftemperature, light and humidity (data not shown). The phenotypes of theNA and LA plants in the greenhouse at harvest (30 days post-topping)were similar to their counterparts grown in the field in terms of plantheight, leaf number and leaf morphology (data not shown). The impact ofwounding on polyamine biosynthesis was minimized by designing theexperiments so that each leaf/root sample was collected only once perplant and time point. Time-course monitoring of the total polyaminecontent in leaves at the same developmental stage—i.e. leaf 12 beforeflowering, leaf 19 at topping and leaf 24 at harvest—revealedsignificantly (p<0.05) higher levels of polyamines in the LA plantsbefore flowering (1.5-fold) and at harvest (2.1-fold) compared to the NAcontrols (FIG. 2B). The LA plants also accumulated significantly(p<0.05) higher levels of total polyamines in the roots at topping(2.4-fold) and at harvest (1.4-fold) compared to the NA controls (FIG.2B)

Example 9: Effect of the Nic1nic2 Double Mutation on PolyamineBiosynthesis

Comparative analysis of the polyamine composition in selected leaves(leaf 6 before flowering, young leaf 23 at topping and mature leaf 23 atharvest) in the four varieties revealed that, before flowering, lines LIand LA contained significantly (p<0.05) higher levels of free putrescinethan the NA controls (1.6-fold and 4.2-fold higher, respectively) andeven higher levels of conjugated putrescine (2.1-fold and 5-fold higher,respectively) (FIG. 3A). The conjugated putrescine and spermidinefractions increased continuously during ripening in all four varieties,but remained significantly higher in LI and LA plants compared to NAcontrols (FIG. 3A). The greatest differential in polyamine content wasobserved in the LA leaves at harvest, with a 2.1-fold increase in thelevel of total polyamines compared to NA controls, including a 1.8-foldincrease in free putrescine, a 2.9-fold increase in conjugatedputrescine and a 2.4-fold increase in conjugated spermidine. However,there was no significant difference between the NA and HI varieties,indicating that the nic2 single mutation had a lower impact on polyamineaccumulation.

At topping, the roots of the LA plants contained significantly (p<0.05)higher levels of free putrescine, conjugated putrescine and conjugatedspermidine than the NA plants (2.6-fold, 2.9-fold and 2.5-foldincreases, respectively) and such differences were also observed atharvest (1.6-fold, 1.4-fold and 2.5-fold increases, respectively) (FIG.3B).

Example 10: The Polyamine Biosynthesis Pathway is More Active in the LAPlants

The relative contribution of ADC and ODC to putrescine biosynthesis wasevaluated by measuring the activity of each enzyme in the leaves (leaf23) and roots of the NA and LA plants at topping and harvest. ADC andODC activity varied in an organ-specific and developmentalstage-specific manner in both lines (FIG. 4 ). Whereas ADC activity washigh in the leaves but minimal in the roots of both lines, ODC activitywas higher in the younger leaves and roots, indicating that ODC ismainly responsible for putrescine biosynthesis in the roots. ADCactivity was significantly higher (1.4-fold, p<0.05) in the leaves ofthe LA plants compared to the NA controls at topping and harvest (FIG.4A). Similarly, ODC activity was significantly higher (p<0.05) in the LAplants compared to the NA controls in the roots at topping (1.8-fold)and at harvest (1.7-fold), and in young leaves at topping (1.5-fold)(FIG. 4B).

Example 11: Inhibition of Polyamine Biosynthesis in the LA Variety

Given the correlation between the higher polyamine levels in the LAvariety and the undesirable leaf morphology, the effect of treating theplants with chemicals that inhibit ADC and ODC was evaluated.Preliminary experiments defined the appropriate inhibitor concentration,application time, treatment intensity and duration (data not shown). Thelevels of free and conjugated putrescine were significantly higher inthe LA plants than the NA controls before flowering and at harvest (FIG.3 ), so the ADC inhibitor D-arginine and the ODC inhibitordifluoromethylornithine (DFMO) were applied beginning 2.5 weeks beforetopping and continued the treatment until harvest. In addition, theplant growth regulator Ethephon® was used alone or in combination withDFMO to accelerate ripening via the liberation of ethylene. To avoid theearly induction of senescence, Ethephon® was applied from topping untilharvest.

The DFMO and DFMO/Ethephon® treatments achieved a partial ameliorationof the morphological phenotype, such that the leaves of the LA plantstook on some of the characteristics of the NA leaves (wrinkling andchlorophyll degradation), whereas treatment with Ethephon® alone reducedthe chlorophyll content but did not affect leaf morphology (FIG. 5 ).Starting the DFMO treatment before flowering resulted in growth arrest,which was not observed when the treatment was started at topping (datanot shown). The D-arginine treatment had no effect on the chlorophylllevel or morphology of the LA plants.

The analysis of polyamine levels revealed that the DFMO treatment 2.5weeks before topping increased the levels of total polyamines in the LAleaves by 2.1-fold, mainly reflecting higher levels of conjugatedputrescine and conjugated spermidine (FIG. 6A). This higher proportionof conjugated polyamines remained until harvest in the plants treatedwith DFMO and DFMO/Ethephon®. In contrast, the treatment with Ethephon®alone led to a significant reduction in total polyamine levels atharvest, mainly reflecting the reduction of free and conjugatedputrescine. In the roots, the DFMO treatment significantly reduced(p<0.05) the total polyamine content of the LA plants at topping(1.5-fold) and at harvest (1.4-fold) due mainly to reduction of free andconjugated putrescine and conjugated spermidine (FIG. 6B). This decreasewas not reversed by the addition of Ethephon®. In contrast to the effectin leaves, the application of Ethephon® alone had no effect on thepolyamine content of the roots. The D-arginine treatment had no effecton the polyamine content of the LA plants. The loss of polyamines in theroots could therefore reflect the inhibition of ODC activity, the mainenzyme responsible for putrescine biosynthesis in roots.

Example 12: Alteration of Polyamine Levels by Genetic Engineering

Modified tobacco plants are made to suppress ODC activity in a nic1 nic2mutant background. A topping-responsive promoter (e.g., SED ID Nos: 1 to21) is used to drive an ODC RNAi cassette (e.g., SEQ ID No: 22) toachieve the suppression of one or more ODC genes (e.g., coding sequencesor protein sequences shown in SEQ ID Nos: 23 to 34). Transgenic plantsare generated and assessed for leaf phenotypes, including for example,total leaf polyamine level, total root polyamine level, total leafchlorophyll level, mesophyll cell number per leaf area unit, leafepidermal cell size, and cured leaf grade.

Modified tobacco plants are also made to modulate the expression andactivity of a MYB8 gene in a nic1 nic2 mutant Burley background. MYB8was reported to control inducible phenolamide levels by activating threehydroxycinnamoyl-coenzyme A:polyamine transferases in Nicotianaattenuata. See Onkokesung et al., Plant Physiology 158 (1) 389-407(2012). A constitutive promoter or a topping-responsive promoter (e.g.,SED ID Nos: 1 to 21) is used to drive an MYB8 RNAi cassette or an MYB8cDNA sequence to achieve suppression or overexpression, respectively.Transgenic plants are generated and assessed for leaf phenotypes,including for example, total leaf polyamine level, total root polyaminelevel, total leaf chlorophyll level, mesophyll cell number per leaf areaunit, leaf epidermal cell size, and cured leaf grade.

Example 13: A Breeding Population

Low-alkaloid tobacco hybrids, varieties, or lines can be made as aBurley type, a dark type, a flue-cured type, a Maryland type or anOriental type tobacco, or can be essentially derived from BU 64, CC 101,CC 200, CC 27, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900,Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpaotobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, K 149,K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, LittleCrittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6,NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal SmithMadole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50,PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70,Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC,TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309,or VA359, Maryland 609, HB3307PLC, HB4488PLC, KT206LC, KT209LC, KT210LC,KT212LC, R610LC, PVH2310, NC196, KTD14LC, KTD6LC, KTD8LC, PD7302LC,PD7305LC, PD7309LC, PD7318LC, PD7319LC, PD7312LC, ShireyLC, or anycommercial tobacco variety according to standard tobacco breedingtechniques known in the art.

The invention claimed is:
 1. A recombinant DNA construct comprising atopping inducible promoter operably linked to a transcribable DNAsequence encoding a non-coding RNA for suppression of an ornithinedecarboxylase (ODC) gene having at least 90% identity to a sequenceselected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27,and
 28. 2. The recombinant DNA construct of claim 1, wherein saidornithine decarboxylase (ODC) gene encodes a polypeptide sequence havingat least 90% identity to a sequence selected from the group consistingof SEQ ID NOs: 29, 30, 31, 32, 33, and
 34. 3. The recombinant DNAconstruct of claim 1, wherein said ODC gene comprises a nucleotidesequence having at least 95% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 23, 24, 25, 26, 27, and
 28. 4. Therecombinant DNA construct of claim 1, wherein said inducible promotercomprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,and
 21. 5. The recombinant DNA construct of claim 1, wherein saidnon-coding RNA comprises a nucleotide sequence having at least 95%identity to a sequence selected from the group consisting of SEQ ID NOs:35 and
 36. 6. The recombinant DNA construct of claim 1, wherein saidnon-coding RNA is provided in an ODC RNAi construct comprising anucleotide sequence having at least 95% identity to SEQ ID NO:
 22. 7.The recombinant DNA construct of claim 1, wherein said non-coding RNA isprovided in an ODC RNAi construct comprising a nucleotide sequencehaving at least 99% identity to SEQ ID NO:
 22. 8. The recombinant DNAconstruct of claim 1, wherein said ornithine decarboxylase (ODC) geneencodes a polypeptide sequence having at least 95% identity to asequence selected from the group consisting of SEQ ID NOs: 29, 30, 31,32, 33, and
 34. 9. The recombinant DNA construct of claim 1, whereinsaid ODC gene comprises a nucleotide sequence having at least 99%identity to a sequence selected from the group consisting of SEQ ID NOs:23, 24, 25, 26, 27, and
 28. 10. The recombinant DNA construct of claim1, wherein said non-coding RNA comprises a nucleotide sequence having atleast 99% identity to a sequence selected from the group consisting ofSEQ ID NOs: 35 and
 36. 11. A recombinant DNA construct comprising atopping inducible promoter having at least 95% identity to a sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, operablylinked to a transcribable DNA sequence encoding a non-coding RNA forsuppression of an ornithine decarboxylase (ODC) gene having at least 90%identity to a sequence selected from the group consisting of SEQ ID NOs:23, 24, 25, 26, 27, and
 28. 12. The recombinant DNA construct of claim1, wherein said ornithine decarboxylase (ODC) gene encodes a polypeptidesequence having at least 90% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 29, 30, 31, 32, 33, and
 34. 13. Therecombinant DNA construct of claim 11, wherein said ODC gene comprises anucleotide sequence having at least 95% identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, and
 28. 14.The recombinant DNA construct of claim 11, wherein said non-coding RNAcomprises a nucleotide sequence having at least 95% identity to asequence selected from the group consisting of SEQ ID NOs: 35 and 36.15. The recombinant DNA construct of claim 11, wherein said ornithinedecarboxylase (ODC) gene encodes a polypeptide sequence having at least95% identity to a sequence selected from the group consisting of SEQ IDNOs: 29, 30, 31, 32, 33, and
 34. 16. The recombinant DNA construct ofclaim 11, wherein said ODC gene comprises a nucleotide sequence havingat least 99% identity to a sequence selected from the group consistingof SEQ ID NOs: 23, 24, 25, 26, 27, and
 28. 17. The recombinant DNAconstruct of claim 11, wherein said non-coding RNA comprises anucleotide sequence having at least 99% identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 35 and 36.